Pancreas: islets of Langerhans
STRUCTURE AND FUNCTION
The pancreas is an elongate, mixed exocrine and endocrine gland that lies adjacent to the duodenum; it is divided into head, body and tail regions.

The exocrine pancreas, the larger component (98-99%), is composed of tubulo-acinar glands that empty, via a highly branched duct system, into the main pancreatic duct. This duct runs the entire length of the gland and opens into the duodenum through the ampulla of Vater. The acinar cells produce digestive enzymes, and some duct lining cells yield fluid rich in sodium and bicarbonate¹.

The endocrine pancreas (1 to 2% of the adult pancreatic mass²) comprises the islets of Langerhans and some scattered cells that occur singly or in small groups³. These are distributed throughout the exocrine pancreas, although the islets are more numerous in the tail1. Endocrine cells are also found within the ductal system³. In haematoxylin and eosin stained sections the islets appear as rounded, compact groups of pale staining cells, with a rich blood supply. However, some islets in the posterior region of the pancreatic head are more irregular in shape, with large elongate cells arranged in trabeculae².

Human islet cells have been classified as four types based on their hormone production: A (glucagon); B (insulin); D (somatostatin); and PP (pancreatic polypeptide)^4,5. The relative frequency and distribution of the cell types within islets are given in Table 1. In the special islets of the pancreatic head the proportion of PP cells is increased² whilst the endocrine cells found outside the islets include both PP and D cells³. Two other cell types occur infrequently: D1 cells that secrete vasoactive intestinal peptide (VIP) and enterochromaffin cells that secrete serotonin⁵.

PATHOLOGY
The most common significant clinical disease of the endocrine pancreas is diabetes mellitus. Associated morphological changes in the islets are not a consistent nor diagnostic feature of diabetes but may include: variation in the size and number of islets; variation in the A:B cell ratio; B cell degranulation; fibrosis; amyloid deposition; and leucocytic infiltration⁵. Cell numbers and the A:B cell ratio can vary in chronic pancreatitis and some other clinical and experimental conditions³.

The most important lesions in terms of morphological and histochemical examination of the endocrine pancreas are the relatively uncommon islet cell tumours. These can arise in any region of the pancreas as single or multiple lesions composed of one or more cell type. Such tumours can be benign or malignant and may produce hormones of pancreatic or non-pancreatic origin at significant levels. The most common presentations are associated with:

· ß-cell tumour (insulinoma) - hyperinsulinism and hypoglycaemia;
· gastrinoma - Zollinger-Ellison syndrome; and
· multiple endocrine neoplasia - including tumours or hyperplasias of other endocrine glands.

A-cell tumours (glucagonomas), D-cell tumours (somatostatinomas), VIP-omas, pancreatic carcinoid tumours and PP-cell tumours are rare⁵. Islet cell tumours which produce more than one hormone are categorised according to the predominant hormone⁶. However, this classification does not always correlate with the clinical symptoms, and the secretory pattern of multihormonal tumours can sometimes change with time⁷ or following chemotherapy³. Since these tumours can also produce hormones of non-pancreatic type, accurate identification of cells and their hormone production is important to determine appropriate therapy and follow-up investigations⁶.

SPECIMEN PREPARATION
To obtain accurate information on the structure and function of the endocrine pancreas multiple samples must be taken from different regions because of the variable distribution of cell types in both normal and pathological conditions⁴. When this is not possible accurate documentation as to the site of the biopsy is required².

Rapid fixation preserves the hormone content and in-situ localisation, but if delay is unavoidable the tissue should be held at 4°C. Bouin's fluid is the preferred fixative for routine haematoxylin and eosin, histochemical and immunocytochemical techniques4 but 10% neutral buffered formalin is also suitable.

STAINING
The different cell types of the endocrine pancreas cannot be distinguished in haematoxylin and eosin stained sections, but a number of histochemical methods allow some differentiation. Unfortunately, these techniques are not sufficiently sensitive, specific nor reliable to use in the definitive classification of islet cell tumours as they do not provide information on the hormone production of the cells. However, histochemical techniques are useful for the initial examination, to demonstrate cell types (primarily A and B cells) and to aid selection of antisera for immunocytochemical studies⁴. The most appropriate histochemical methods are Gomori's aldehyde fuchsin (AF) for B cells and the Grimelius argyrophil technique for A and other endocrine cells⁴. Immunocytochemical techniques, using specific antisera against the various hormones produced by islet cell tumours, are required to accurately determine the functional capacity of these tumours. Other useful techniques include: chrome alum haematoxylin-phloxine for A and B cells⁸; phosphotungstic acid haematoxylin for A cells⁹; lead haematoxylin for A, D and other endocrine cells⁸ and; combined argyrophil, AF and lead haematoxylin for demonstration of A, B and D cells in a single section¹⁰. The different cell types can also be identified according to the ultrastructural morphology of their secretory granules¹¹.

GOMORI'S ALDEHYDE FUCHSIN
In 1950 Gomori¹² described an 'aldehyde fuchsin' staining technique that demonstrated elastic fibres, mast cell granules, gastric chief cells, pancreatic islet B cells, some basophils of the anterior pituitary and some mucins. Despite numerous investigations the mechanism of AF staining of pancreatic islet B-cell granules has not been conclusively determined,^13,14,15 but differs between oxidised and unoxidised sections. Mowry^16,17 detailed three types of AF reactions:

· staining of sulphated and carboxylated tissue polyanions (mast cell granules, acidic mucins) as occurs with other basic (cationic) dyes, such as Alcian Blue;
· staining of polyaldehydes produced in tissue by oxidation of poly-vic-glycols (glycogen, epithelial mucins);
· reaction with certain non-ionic proteins and polypeptides rich in cystine, without prior oxidation (pancreatic islet B-cell granules, elastic fibres).

This third reaction type describes the unique property of AF solutions when applied to unoxidised tissue sections; prestaining of tissue polyanions with Alcian Blue has been advocated to increase the selectivity of the subsequent AF staining^16,17 AF is thought to bind to the membrane of the B-cell granule rather than the granule contents (insulin and proinsulin) in unoxidised sections^18,19,20. In oxidised sections the cystine-rich substances are rendered acidic (anionic) and stain with basic (cationic) dyes¹⁶.

A number of factors affect the staining of pancreatic B-cell granules with AF^12,16,17,21:

Fixation
Fixation must be prompt and sufficient to prevent dissolution of the insulin-containing B-cell granules in the acid-ethanol solvent of the AF solution. Thorough fixation (at least 24 hours) in formalin or Bouin's solution is recommended as these fixatives provide a clear background. Mercury-containing fixatives produce a pale lilac background, whilst those containing dichromate produce murky staining and should be avoided.

Oxidation
Gomori noted that more rapid and intense staining was obtained with AF after iodine/sulphite treatment. Scott²² used permanganate (a stronger oxidant) to decrease staining times further and to increase staining intensity, but selectivity is also decreased as other tissue components are altered by oxidation. Without prior oxidation staining times of up to 4 hours may be required to achieve comparable results, depending upon the age of the AF solution.

Basic fuchsin
This is a mixture of Pararosaniline, Rosaniline and other dye homologues. The Pararosaniline component (CI 42500) is responsible for staining pancreatic B-cell granules in unoxidised sections.

The aldehyde
Gomori found, after using different aldehydes when preparing AF, that paraldehyde gave the best results. A more recent study²³ has concluded that only paraldehyde and acetaldehyde are effective for AF staining of unoxidised pancreatic B-cell granules. Paraldehyde must be fresh and is best obtained in small volumes or aliquoted and stored at 4°C. Some protocols use 3% paraldehyde which reportedly gives stronger staining.

Ripening of AF
It is widely accepted that the ripening of AF at room temperature requires at least two to three days for useful staining and up to five days to eliminate all background staining. The time may be decreased by heating the solution.

Shelf life of AF
The useful life of AF solutions is prolonged by storage at 4°C, but the preparation should be brought to room temperature before staining. The precipitate that forms during storage can be removed by rapid filtration or centrifugation, although repeated treatments will decrease the potency of the staining solution. The capacity for staining elastic fibres is retained longer than pancreatic B-cell granules. If AF staining is performed routinely, variability is reduced by maintaining a fresh solution that should be replaced regularly (monthly)²⁴. Otherwise as the solution ages, staining times may need to be increased. Dried powder forms of AF that are reconstituted for use give inferior staining results to those obtained with conventionally prepared AF solutions^14,21.

Modified Gomori aldehyde fuchsin²⁵
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in Bouin's fluid or 10% neutral buffered formalin. Pancreas is recommended as control tissue.

REAGENTS REQUIRED
1 Lugol's iodine
2 5% aqueous sodium thiosulphate
Sodium thiosulphate 5 g
Distilled water 100 ml
3 Aldehyde fuchsin
Pararosaniline (CI 42500) 0.5 g
70% ethanol 100 ml
Paraldehyde 1 ml
Concentrated hydrochloric acid 1 ml
Dissolve the pararosaniline in the ethanol. Add the paraldehyde and hydrochloric acid. Allow to ripen at room temperature in the dark for 3 to 5 days then store at 4°C.
4 Light green/orange G
Light Green SF Yellowish (CI 42095) 0.2 g
Orange G (CI 16230) 1 g
Phosphotungstic acid 0.5 g
Distilled water 100 ml
Glacial acetic acid 1 ml
Mix in the proportions indicated. This stain keeps well at room temperature.
5 Celestin blue/alum haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Place sections in Lugol's iodine for 30 minutes.
3 Wash in water.
4 Decolourise with sodium thiosulphate for 2 minutes.
5 Wash in water.
6 Rinse in 70% ethanol.
7 Immerse sections in aldehyde fuchsin staining solution for 15-30 minutes. Check staining microscopically (see note 3).
8 Rinse in 95% ethanol.
9 Wash in water.
10 Stain nuclei with celestine blue/alum haematoxylin, differentiate and blue.
11 Rinse in distilled water.
12 Counterstain with light green/orange G for 45 seconds.
13 Rinse briefly in 0.2% acetic acid, then in 95% ethanol.
14 Dehydrate, clear and mount.

RESULT
Nuclei - blue/black
B-cell granules - purple
A-cell granules - yellow
D-cell granules - green
Collagen - green
Mast cells, elastic fibres and some mucins - purple

TECHNICAL NOTES
1 For direct AF staining (no oxidation) omit steps 2 to 5 (above) and increase the AF staining time (step 7) as necessary.
2 When using Scott's permanganate oxidation replace the iodine/thiosulphate treatment (steps 2 to 5, above) as follows:
Step 2. Oxidise sections in a mixture of equal parts of 0.5% potassium permanganate and 0.5% sulphuric acid for 2 minutes.
Step 3. Rinse in water.
Step 4. Decolourise in 2% sodium bisulphite until the section appears white.
Step 5. Wash well in water for 2 minutes.
3 AF staining must be monitored microscopically as it progresses. Rinse slides in 95% ethanol, examine microscopically and replace in the staining solution until the desired staining is obtained.
4 Alternative counterstains include: the Grimelius argyrophil technique^17,26,27 and Gomori's phloxine²⁸.

THE GRIMELIUS ARGYROPHIL TECHNIQUE²⁶
In 1968 Grimelius described an argyrophil technique to demonstrate A (or a ₂)²⁹ cells of the pancreatic islets, in formalin or Bouin's fixed tissue. This technique can be used as a general method for endocrine cells (particularly those found scattered through the exocrine pancreas²⁶ and in the gastrointestinal tract²⁷) and is of value in identifying endocrine tumours that show atypical morphology on light microscopy⁷. The glucagon component of A-cell granules is not demonstrated³⁰ but rather the argyrophilia corresponds to the presence of chromogranin A, a granular protein³¹. The B and D cells of the endocrine pancreas are not demonstrated by this method⁴. The original technique (variant II)^26,27 requires silver impregnation at 60°C for 3 hours, but this time can be reduced using microwave irradiation (15 minutes)^32,33 or by preheating the silver and reducing solutions (10 minutes)³⁴. A one-step combined silver and reducing solution method (25 minutes, microwave 5 minutes) has also been described³⁵.

SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral buffered formalin or Bouin's fluid. Fixatives containing ethanol, mercuric chloride, dichromate or osmium should not be used³⁶. Pancreas is recommended as control tissue.

REAGENT PREPARATION
1 0.2 mol/l acetate buffer, pH 5.6
a) 1.2% acetic acid
Glacial acetic acid 1.2 ml
Distilled water 100 ml
b) 1.64% sodium acetate
Sodium acetate anhydrous 1.64 g
Distilled water 100 ml
Mix 4.8 ml of solution a) and 45.2 ml of solution b).
2 Silver nitrate solution
0.2 mol/l acetate buffer, pH 5.6 10 ml
Distilled water 87 ml
1% aqueous silver nitrate 3 ml
3 Reducing solution
Hydroquinone 1 g
Sodium sulphite 5 g
Distilled water 100 ml
4 Kernechtrot
Kernechtrot (Nuclear Fast Red CI 60760) 0.1 g
Aluminium sulphate 5 g
Distilled water 100 ml
Dissolve with heat. Cool and filter.

METHOD
1 Dewax and rehydrate sections.
2 Place slides in silver nitrate solution at room temperature and then incubate at 60°C for 3 hours.
3 Drain slides and place in freshly prepared reducing solution at 40-45°C for 1 minute.
4 Rinse in distilled water.
Note: A weak argyrophil reaction can be enhanced by double impregnation:
a) Place slides in 5% sodium thiosulphate for 2 minutes.
b) Wash slides in distilled water for 5 minutes.
c) Place in freshly prepared silver solution at room temperature for 15 minutes.
d) Drain slides and place in freshly prepared reducing solution at 55°C for 1 minute.
e) Rinse in distilled water.
5 Counterstain nuclei with Kernechtrot, if desired.
6 Rinse rapidly in distilled water.
7 Dehydrate, clear and mount.

RESULTS
Argyrophil granules - black
Nuclei - red

TECHNICAL NOTES
1 To intensify the reaction when demonstrating argyrophil cells of the gastrointestinal tract increase the silver nitrate concentration to 0.07% and the temperature of the reducing solution to 55°C²⁷.
2 Distilled water rinses between the impregnation and reducing solutions reduce the non-specific yellow background staining³⁴.
3 The yellow background staining usually enables tissue orientation.

Pituitary
The pituitary gland (or hypophysis) lies centrally at the base of the brain in a depression in the floor of the cranial cavity. It is attached to the hypothalamus via the infundibulum and consists of two main lobes, designated anterior and posterior according to their anatomical positions. These two lobes are developmentally, structurally and functionally distinct.

Anterior pituitary
STRUCTURE AND FUNCTION
The anterior lobe (or adenohypophysis) is the glandular portion, composed of nests and cords of cells within an interlacing network of small capillaries. The cells of the anterior pituitary produce growth hormone (GH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), melanocyte stimulating hormone (MSH), b -lipotropin (b -LPH), follicle stimulating hormone (FSH), luteinising or interstitial cell stimulating hormone (LH or ICSH) and thyroid stimulating hormone (TSH). Secretion is regulated by 'releasing' and 'inhibiting' factors produced in the hypothalamus and transported to the anterior lobe via the hypophyseal portal veins. A negative feedback system controls the secretion of these factors³⁷.

SPECIMEN PREPARATION
Pituitary tissue must be fixed promptly to prevent the diffusion and/or loss of hormones, a problem that occurs when interruption to the blood supply is prolonged or the gland is traumatised during removal³⁸. Post mortem specimens should be collected within 4 hours of death³⁹. Fixation in 10% neutral buffered formalin preserves morphological detail and retains hormones in situ, but for specific staining techniques other fixatives are recommended^40,41,42.

The gland should be sectioned in the horizontal plane for optimal examination of the medial 'mucoid' wedge and lateral 'acidophil' wings of the anterior lobe. This enables evaluation of the distribution and relative frequency of the different cell types³⁸.

STAINING
Three cell types (acidophils, basophils and chromophobes) can be identified in thin haematoxylin and eosin stained sections as their respective secretory granules have differing affinities for acidic and basic dyes^1,43 (Table 1) although some simple special stains will give improved cellular differentiation (Table 2). Other techniques allow basophils to be separated into two subtypes (Table 3), and when this information is combined with morphological criteria and the pattern of cell distribution, three or more subtypes can be identified. Unfortunately, some of these methods are complex and require experience to obtain good quality, consistent staining results. Those which use a strong oxidising agent (especially performic acid) can also disrupt tissue and result in section loss⁴². Acidophils can be divided into two subtypes by selective staining of prolactin producing cells⁴⁸ using either Herlant's erythrocin stain41 or Brooks' carmoisine technique⁴⁴. The orange G-acid fuchsin-light green (OFG) method of Slidders⁴⁰ is a suitable routine stain for the differentiation of acidophils, basophils and chromophobes, and the architectural pattern of the gland is well demonstrated with a reticulin stain⁴³. A control section of normal pituitary should always be stained in parallel with the test case to allow comparison with normal morphology and cell distribution³⁸.

Immunocytochemical techniques using specific antisera to pituitary hormones have enabled the identification of five distinct cell types in the anterior lobe^48,49 (Table 4). Studies comparing conventional dye techniques with immunocytochemical localisation of pituitary hormones show that the conventional methods do not reliably differentiate the various cell types present^47,48,50,51. Some anterior pituitary cells contain more than one hormone and chromophobes, previously thought to be resting acidophils or basophils, or transitory cells, may contain hormones. In addition, cells containing the same hormone can stain variably with dye techniques³⁸. Such discrepancies can be attributed to the staining of substances other than hormones⁵¹ or may reflect different stages in cell maturation or the cell secretory cycle⁴⁷.

Ultrastructural features of the different cell types and their granules^11,43 are not sufficiently distinct to allow definitive cell identification1 but are of value when used in conjunction with immunocytochemistry¹¹. It should be noted that there is significant inter-species variation in the structure of the pituitary gland and information from animal studies cannot be extrapolated to humans⁴⁸.

PATHOLOGY
Variations in the number, size and distribution of the different cell types in the anterior pituitary are seen in a number of conditions. During pregnancy and lactation the gland may increase in size by up to one third due to a true hyperplasia of prolactin secreting cells. Changes in other cell types occur in some endocrinopathies, following surgical removal of pituitary hormone target organs, and in conjunction with certain drug therapies. The gland also shows age related changes^43,48. The most significant lesions seen in the pituitary gland are benign adenomas^37,38. Current classifications identify ten types of pituitary adenoma based on immunocytochemical demonstration of specific hormone production and tumour cell ultrastructure^48,49. Tumour cells may be non-secretory or produce one or more hormones: chromophobic adenomas (the most common type) are able to produce any of the anterior pituitary hormones; acidophilic adenomas produce PRL and/or GH; and basophilic adenomas produce ACTH, b -LPH and/or endorphins⁴⁸. TSH, LH or FSH secreting adenomas are uncommon³⁷.

Orange G - Acid Fuchsin - Light Green⁴⁰
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in mercury-containing fixatives or 10% neutral buffered formalin. A known positive control (pituitary) should be included.

REAGENT PREPARATION
1 Orange G solution
G (CI 16230) 0.5 g
Absolute ethanol 95 ml
Distilled water 5 ml
Phosphotungstic acid 2 g

2 Acid fuchsin solution
Acid Fuchsin (CI 42685) 0.5 g
Distilled water 99.5 ml
Glacial acetic acid 0.5 ml

3 1% phosphotungstic acid
Phosphotungstic acid 1 g
Distilled water 100 ml

4 Light green solution
Light Green (CI 42095) 1.5 g
Distilled water 98 ml
Glacial acetic acid 2 ml

5 Celestine blue/alum haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Remove mercury pigment with iodine/thiosulphate.
3 Stain nuclei with celestine blue/alum haematoxylin.
4 Rinse in 95% ethanol.
5 Stain sections with orange G solution for 2 minutes.
6 Rinse in distilled water.
7 Stain sections with acid fuchsin solution for 2-5 minutes. Staining is progressive and should be continued until the basophils, but not the background, are strongly stained.
8 Rinse in water.
9 Treat sections with 1% phosphotungstic acid for 5 minutes.
10 Rinse in water.
11 Stain sections with light green solution for 1-2 minutes.
12 Rinse in water.
13 Dehydrate, clear and mount.

RESULTS
Nuclei - blue/black
Acidophils - orange/yellow
Basophils - magenta red
Chromophobes - pale grey/green
Erythrocytes - ;yellow
Stroma - green

TECHNICAL NOTES
1 If tissue has been fixed in formalin, staining can be enhanced by mordanting sections in picro-mercuric-alcohol (a saturated solution of picric acid in absolute alcohol containing 3% mercuric chloride) overnight. This solution is particularly toxic and care is required when preparing or handling it. Subsequently, mercury pigment must be removed with iodine/thiosulphate and the sections washed well in water to remove picric acid staining. Helly's or Bouin's fluids can also be used.
2 Iodine/thiosulphate treatment is recommended, even when mercury-containing solutions have not been used, to enhance staining.

Posterior pituitary
The posterior lobe (or neurohypophysis) is the neural portion containing pituicytes (specialised supportive cells similar to the neuroglial cells of the central nervous system) and unmyelinated nerve axons that originate from neurosecretory cells located in the hypothalamus. The posterior lobe stores and secretes two hormones, oxytocin and vasopressin (anti-diuretic hormone). These are produced by the neuronal cell bodies in the hypothalamus, transported to the posterior lobe along the axons by carrier proteins (neurophysins) and stored as neurosecretory granules (Herring bodies) at the nerve terminals1.

The nerve axons can be demonstrated using silver impregnation techniques. Neurosecretory substances stain blue to purple with Alcian Blue or Aldehyde Thionin in the following anterior pituitary staining methods: permanganate-aldehyde thionin-periodic acid-Schiff-orange G (PM-AT-PAS-OG)⁴⁶, bromine-alcian blue-orange G-acid fuchsin-light green (Br-AB-OFG)⁸ or; Bargmann's modification of Gomori's chrome haematoxylin⁴⁵. Immunocytochemical techniques using specific antisera can demonstrate the hormones secreted (oxytocin, vasopressin), the neural origin of the tissue (S100 protein, neurone specific enolase) and the glial nature of pituicytes (glial fibrillary acidic protein).

Disorders of the posterior pituitary are very rare and usually result in vasopressin deficiency or inappropriate release of vasopressin³⁷.

Between the two main lobes lies the intermediate lobe (a poorly defined region in humans) that contains cystic structures lined by epithelium and filled with colloidal material (remnants of the embryonal Rathke's pouch)1. In other species (amphibians) the cells of the intermediate lobe secrete MSH but in humans the function of these cells is uncertain⁵².

Paneth cells
STRUCTURE AND FUNCTION
Paneth cells are part of the epithelial lining of the human small intestine but are present only in the base of the crypts of Lieberkühn. Although the precise function of Paneth cells is unclear, lysozyme has been described in the secretory granules, Golgi apparatus and rER53, and immunoglobulins (IgA and IgG) have been demonstrated in the cell cytoplasm⁵⁴. The presence of lysozyme (an enzyme with limited bactericidal activity that is enhanced in the presence of immunoglobulins and complement) combined with the phagocytic capability of Paneth cells, suggests they are involved in regulation of the microbial flora of the small intestine1. Cationic trypsin immunoreactivity has been demonstrated in human Paneth cells, indicating a possible role in secretion of digestive enzymes⁵⁵, and their content of heavy metals, particularly zinc, suggests a role in the elimination of metals⁵⁶.

PATHOLOGY
The response of Paneth cells in pathological conditions is variable. Cell numbers may be decreased due to non-specific injury in inflammatory conditions and increased in regions undergoing regeneration and repair. Reports of alterations in coeliac disease are inconsistent⁵⁶. Characteristic inclusion bodies are seen (by electron microscopy) in Paneth cells in acrodermatitis enteropathica, a rare autosomal recessive disease characterised by primary zinc deficiency. Following dietary zinc supplementation these inclusions disappear⁵⁷. Similar inclusions have been reported in neoplastic Paneth cells⁵⁸.

Metaplastic Paneth cells have been demonstrated in other parts of the gastrointestinal tract in Barrett's oesophagus, chronic gastritis, various inflammatory conditions of the large intestine and in association with some tumours. The presence of metaplastic Paneth cells is thought to represent a non-specific response to the disease state (their evolution and function in these sites is unknown)⁵⁹.

SPECIMEN PREPARATION
Tissue fixation must be prompt as Paneth cells degranulate rapidly during autolysis. The granules dissolve in fixatives containing acetic acid but are well preserved in 10% neutral buffered formalin or mercuric chloride-formalin (formol sublimate)^56,60.

STAINING
Paneth cells are recognised in haematoxylin and eosin stained sections by their large, intensely eosinophilic apical secretory granules and their localisation in the base of the crypts of Lieberkühn. They can also be demonstrated using Lendrum's phloxine tartrazine⁶⁰, Masson's trichrome (granules stain red with both methods)⁶¹ and Mallory's phosphotungstic acid haematoxylin (granules stain blue/black)^59,62. The granules stain variably (weakly) with the PAS technique^61,62 and react histochemically for tryptophan, tyrosine and sulphydryl and disulphide groups⁶³.

Lendrum's phloxine tartrazine⁶⁰ is a simple and reliable technique. Nuclei are stained with haematoxylin, followed by cytoplasmic staining with phloxine and subsequent differentiation in a solution of tartrazine in cellosolve. As differentiation continues the red phloxine staining is progressively removed from tissue components that then stain with the yellow tartrazine dye. Paneth cell granules have a strong affinity for phloxine and therefore retain their red colour even after lengthy differentiation. Dye techniques are adequate for demonstrating Paneth cells in sections but immunocytochemical techniques are required to identify specific substances contained within Paneth cells (lysozyme, immunoglobulins, cationic trypsin)^53,54,55.

Lendrum's phloxine tartrazine (Lendrum 1947)⁶⁰
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral buffered formalin or mercuric chloride-formalin. Avoid fixatives containing acetic acid. Small intestine should be used as control tissue.

REAGENT PREPARATION
1 Phloxine solution
Phloxine B (CI 45410) 0.5 g
Calcium chloride 0.5 g
Distilled water 100 ml

2 Tartrazine in cellosolve⁶⁴
Tartrazine (CI 19140) 2.5 g
Cellosolve (ethylene glycol monoethyl ester) 100 ml
This is a saturated solution.

3 Celestine blue/alum haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Stain nuclei with celestine blue/alum haematoxylin.
3 Stain in phloxine solution for 30 minutes.
4 Rinse briefly in water.
5 Differentiate with tartrazine in cellosolve until only the granules stain intensely red (control microscopically).
6 Rinse briefly in water (see note 3).
7 Rinse in 95% ethanol.
8 Dehydrate, clear and mount.

RESULTS
Nuclei - blue
Paneth cell granules - red
Background - yellow

TECHNICAL NOTES
1 Although Lendrum considered the use of an iron haematoxylin unnecessary, it does provide more intense nuclear staining.
2 Calcium chloride added to the phloxine solution intensifies the stain and prolongs the shelf life for up to one year.
3 Lendrum recommended prolonged differentiation followed by thorough washing in water to remove virtually all the yellow tartrazine staining. This facilitates the demonstration of Paneth cells by increasing the contrast. If intense yellow staining is preferred only brief washing is required at step 6.
4 Other tissue components can be demonstrated with this technique by varying the extent of differentiation. These include muscle, fibrin, keratin, viral inclusion bodies, pancreatic B-cell granules and Russell bodies. Appropriate control sections must be used to monitor differentiation.

Seromucous gland cells
seromucous acini are foundin the submandibular and sublingual salivary glands and in subepithelial connective tissue of parts of the upper respiratory tract. In tissue sections, seromucous glands appear s mucous acini with a crescentic cap (demilune) of serous cells. The mucous cells appear pale in H&E stained sections, as the cytoplasmic mucin does not stain or reacts weakly with haematoxylin. both acidic and neutral mucins can be demonstrated in these cells by the alcian blue (pH 2.5) - periodic acid - Schiff (AB-PAS) technique. The apical secretory granules of the serous cells are basophilic, and PAS and PAS-diastase positive.⁶⁵ They are dissolved in fixatives containing acetic acid, but are well preserved in 10% neutral buffered formalin.⁶⁶

Mast cells
STRUCTURE AND FUNCTION
Mast cells are normally present in small numbers in the connective tissue of all organs, but particularly in the dermal layer of skin (around blood vessels and nerves), and are identified by their cytoplasmic granules. Mast cells have been considered the tissue equivalent of the circulating basophil but, while there is evidence that they arise from common precursor cell in the bone marrow, there is no evidence that mature basophils are able to differentiate into mast cells⁶⁷. The two cell types are readily distinguished by their morphology on light microscopy^67,68 and the presence of chloroacetate esterase activity in mast cells⁶⁹.

Mast cells play an important role in immunity, with specific involvement in type I (anaphylactic) hypersensitivity reactions. When IgE antibodies are raised against a particular allergen they can bind to mast cell surface Fc receptors. Subsequent exposure to the allergen triggers mast cell degranulation and the release of chemical mediators, such as histamine and heparin, into the surrounding tissues⁵. Mast cells are also involved in delayed hypersensitivity, cytotoxicity, immunoregulation and inflammation⁷⁰.

The content of mast cell granules varies between species and in some pathological conditions. Human mast cells contain histamine, heparin and various proteins, and although serotonin is not normally present, it has been demonstrated in mast cells in the stroma of carcinoid tumours and in mastocytosis⁶⁸.

A subpopulation of mast cells has been identified at mucosal surfaces, such as in the gastrointestinal and respiratory tracts, in rats and humans. These mucosal mast cells show some structural and functional differences from connective tissue mast cells^68,71 and require special fixation conditions or modified staining protocols for their demonstration^70,72-75.

PATHOLOGY
Increased numbers of mast cells are found in many pathological conditions. Mast cell hyperplasia in the skin (mastocytosis) manifests with skin lesions and may present with symptoms of urticaria and flushing due to the chemical mediators released during mast cell degranulation. Children may develop single mastocytomas or the multiple cutaneous lesions of urticaria pigmentosa. In adults, multiple organ involvement can occur (notably affecting bone, liver, spleen and lymph nodes) even without apparent skin lesions (systemic mastocytosis). Lesions of the bone may be localised or widespread, osteoclastic or osteoblastic⁷⁶. Increased mast cell numbers are also seen in some inflammatory bowel diseases (ulcerative colitis, Crohn's disease) and in parasitic infections⁶⁸. Cutaneous neurofibromas, benign and malignant breast lesions, and some soft tissue tumours also show high numbers of mast cells.

SPECIMEN PREPARATION
Fixation must be rapid to avoid cytoplasmic degranulation and deterioration of granule contents. Neutral buffered formalin (10%) preserves morphology and enables demonstration of connective tissue mast cell granules using a variety of staining techniques. Special fixation is required for the demonstration of mucosal mast cells as aldehydes reversibly block cationic dye-binding sites⁷⁴. Carnoy's fluid (minimum fixation time: two hours) or isotonic-formol-acetic acid (1.5% formalin, 0.5% glacial acetic acid - minimum fixation time: two days) are suitable⁷³. Alternatively, mucosal mast cells can be demonstrated in aldehyde fixed tissue by increasing the staining time⁷⁴.

STAINING
Mast cells are not readily identified in haematoxylin and eosin stained sections (the granules are refractile and do not stain) but are well demonstrated by a number of special staining methods. The most common are metachromatic dye techniques and the demonstration of chloroacetate esterase activity.

METACHROMATIC DYE TECHNIQUES
Metachromatic dyes, such as Toluidine Blue and Azure A, demonstrate the strongly sulphated acid mucopolysaccharide (heparin) content of mast cell granules. The acidified toluidine blue method of Churukian and Schenk⁷⁷ is simple, rapid and reliable. Sections are oxidised with permanganate, decolourised and then stained in an acidified solution of Toluidine Blue. The low pH (3.2) of the solution minimises background and enhances nuclear staining (both nuclear and background staining can be eliminated by lowering the pH to 0.568).

Acidified toluidine blue⁷⁷
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral buffered formalin. Tissue known to contain mast cells (neurofibroma, skin) is used as a control.

REAGENT PREPARATION
1 0.5% aqueous potassium permanganate
Potassium permanganate 0.5 g
Distilled water 100 ml

2 2% aqueous potassium metabisulphite
Potassium metabisulphite 2 g
Distilled water 100 ml

3 Acidified toluidine blue solution (pH 3.2)
Distilled water 99.75 ml
Glacial acetic acid 0.25 ml
Toluidine Blue (CI 52040) 0.02 g

METHOD
1 Dewax and rehydrate sections.
2 Transfer sections to potassium permanganate solution for 2 minutes.
3 Rinse in distilled water.
4 Transfer sections to potassium metabisulphite solution for 1 minute (or until sections appear white).
5 Wash in tap water for 3 minutes.
6 Rinse in distilled water.
7 Place in acidified toluidine blue solution for 5 minutes.
8 Rinse in distilled water.
9 Dehydrate rapidly, clear and mount.

RESULTS
Mast cell granules and other strongly sulphated acid mucopolysaccharides - purple
Nuclei - blue

TECHNICAL NOTE
Mast cells and eosinophils can be demonstrated in the same section by staining with Congo Red before acidified toluidine blue78 and this technique can be modified for undecalcified bone sections⁷⁹.

A prolonged staining protocol⁷⁴ can be used to demonstrate mucosal mast cells in aldehyde fixed tissue, connective tissue mast cells after prolonged aldehyde fixation or the presence of low numbers of mast cells in highly cellular tissue (such as lymph node). Sections are treated with a 0.5% solution of Toluidine Blue in 0.5 mol/l HCl (pH 0.5) for 5-7 days, filtering the solution on alternate days. The mucosal mast cell granules stain dark blue against a clean background. An eosin counterstain (1% eosin in 70% ethanol for 20 seconds) will improve contrast and allow tissue orientation. Eosinophils are demonstrated after counterstaining with eosin at pH 10.

CHLOROACETATE ESTERASE ACTIVITY
Mast cell granules contain proteases, including esterases that rapidly hydrolyse the a -chloroacyl esters of a -naphthol and naphthol AS⁸⁰. Chloroacetate esterase activity is demonstrated by a simultaneous capture technique using the substrate naphthol AS-D chloroacetate and diazonium salts such as pararosaniline, Fast Blue RR or Fast Garnet GBC^81,82,83. Mast cell granules are demonstrated after a short incubation time, before other tissue staining becomes apparent. Leucocytes of the myeloid series are also demonstrated. Pararosaniline is preferred as it forms an insoluble red/pink reaction product and sections can be mounted in synthetic resin. Fast Blue RR forms a vivid blue reaction product and Fast Garnet GBC a red product, but both are soluble in organic solvents and require an aqueous mountant.

Chloroacetate esterase^82,84
SPECIMEN PREPARATION
Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral buffered formalin. Do not use acid-containing fixatives such as Zenker's or Bouin's. Fix smears/imprints for 30 minutes in a solution of 9 parts methanol, 1 part formalin. Wash in tap water and air dry. Tissue known to contain mast cells (or kidney - tubule lining cells; liver - hepatocytes, Kupffer cells) is used as a control.

REAGENT PREPARATION
1 4% pararosaniline in 2 mol/l HCl
Pararosaniline (CI 42500) 0.4 g
Distilled water 8.4 g
Concentrated hydrochloric acid 1.6 ml
Dissolve the pararosaniline in the water. Add the acid slowly.

2 4% aqueous sodium nitrite
Sodium nitrite 0.4 g
Distilled water 10 ml

3 0.07 mol/l phosphate buffer pH 6.5
a) Disodium hydrogen orthophosphate (anhydrous) 9.465 g/l
b) Sodium dihydrogen orthophosphate 10.452 g/l
Mix 30 ml of solution a) and 70 ml of solution b).

4 Substrate solution
Naphthol AS-D chloroacetate 0.01 g
(Store below 0°C)
N-dimethylformamide 1 ml

5 Mayer's haematoxylin

METHOD
1 Dewax and rehydrate sections.
2 Mix 0.1 ml pararosaniline (solution 1), with 0.1 ml of sodium nitrite (solution 2). Leave for 30-60 seconds.
3 Add 30 ml of buffer (solution 3). Check that the solution is at pH 6.3.
4 Add substrate (solution 4), mix and filter.
5 Immediately place sections in the solution and incubate at room temperature for 30 minutes.
6 Check microscopically; if the reaction is incomplete, refilter the solution and reincubate sections for further 15-30 minutes.
7 Wash slides in water for 5 minutes.
8 Counterstain in Mayer's haematoxylin for 5 minutes.
9 Wash in water.
10 Dehydrate, clear and mount.

RESULTS
Esterase activity - red/pink
Nuclei - blue

TECHNICAL NOTES
1 Sections should not be treated with iodine/thiosulphate to remove mercuric pigment as this may reduce the intensity of staining.
2 Do not overheat sections when drying as this destroys enzyme activity.
3 Solutions 1 and 2 keep for at least a month but the reaction solution must be made up fresh each time.
4 If the solution turns red when the buffer is added (step 3) the reaction of pararosaniline with the nitrite was incomplete and a fresh solution should be prepared.

A combination of Human Platelet Factor 4 (HPF 4) which binds to heparin and an anti-HPF 4 antibody can be used to specifically demonstrate mast cell granules in formalin-fixed, paraffin-embedded tissue sections by immunocytochemical techniques⁸⁵. An antiserum directed against histamine has also been used in an indirect immunofluorescent technique on frozen sections. Positive immunostaining in mast cells was only produced in tissue fixed in 4% carbodiimide in O.1 mol/l phosphate buffer (pH 7.4)86. Lectins (000) have been used in studies on the heterogeneity of mast cells⁸⁷.

Romanowsky techniques
HISTORY^88-92
In 1879 Ehrlich described the use of 'neutral' dyes (mixtures of acidic and basic dyes) for the differentiation of cells in peripheral blood smears. In 1891 Romanowsky and Malakowsky independently developed a method which used mixtures of Eosin Y and 'ripened' Methylene Blue that not only differentiated blood cells, but also demonstrated the nuclei of malarial parasites. A number of 'ripening' (oxidation or polychroming) techniques were investigated by different groups (Unna 1891, Nocht 1898) but the aqueous dye solutions produced were unstable and precipitated rapidly. Subsequently, methanol was introduced as a solvent for the dye precipitate (Jenner 1899, England; May and Grünwald 1902, Germany) and techniques were developed that utilised the fixative properties of the methanolic solution, prior to aqueous dilution for staining (Leishman 1901, England; Wright 1902, Germany). Giemsa (1902) further improved these techniques by using more controlled methods of oxidation with measured amounts of known dyes, and adding glycerol to the methanol solvent to increase the solubility and stability of the dyes.

TRADITIONAL FORMULAE AND MECHANISM OF STAINING
Traditional Romanowsky-type dyes are produced by oxidation (polychroming) of Methylene Blue in aqueous solution, using heat and alkali. The resulting solution contains a mixture of Azure A, Azure B, Methylene Violet and Methylene Blue. A measured quantity of Eosin Y is then added to produce a 'neutral' dye. The precipitate formed is dissolved in methanol, or a mixture of equal volumes of methanol and glycerol, to produce a stable stock solution. Working solutions are prepared by diluting the stock solution with distilled water, or an aqueous buffer, to allow ionisation of the dyes. Typical staining results obtained using air-dried, methanol-fixed smears are:

The full range of colours obtained cannot be accounted for by ionic binding alone⁸⁸. The 'red' staining of the nuclei of parasitic protozoa is thought to be produced by the azure dye and eosin binding to the basic protein protamine, rather than the nuclear chromatin, to produce an imino base of the azure dye (which is red). Metachromasia also contributes to the staining pattern observed. Mixtures of pure azure B and eosin Y alone can also produce the same staining pattern as traditional Romanowsky-type dye mixtures⁹³.

CURRENT PRACTICE
Romanowsky-type stains are used routinely in haematology and cytology but are not commonly applied to tissue sections. The examination of smears or imprints and thin (1-2 µm) sections stained with both H&E and a Romanowsky-type stain is recommended for optimal evaluation of bone marrow^94,95 and lymphoid tissues^89,96. Advantages of Romanowsky-type staining over haematoxylin and eosin include: demonstration of mast cell granules; differentiation of mucosubstances; and identification of blood and marrow cells of different lineages and stages of maturation^97,98. Numerous modifications of the Romanowsky method have been described, particularly for application to tissue sections and, although the various techniques may produce slight differences in staining, the basic mechanisms and staining effects are the same. The staining obtained in tissue sections is more variable than in smears because the protocols used require additional steps (differentiation, dehydration, clearing) and tissue sections contain more stainable components⁹⁸. Some stains are used in combination to produce the desired colouring of cell and tissue component as for example with the Jenner-Giemsa and May Grünwald-Giemsa techniques⁹⁹.

SPECIMEN PREPARATION
Bone marrow cores require fixation and decalcification before processing. Zenker's fluid, which simultaneously fixes and decalcifies tissue, is recommended, but formalin fixation followed by mild acid or EDTA decalcification can be used. However, formalin fixation alters the expected staining pattern with the nuclei staining dark blue and the cytoplasm pale blue¹⁰⁰, although the staining of cytoplasmic granules remains unchanged. Decalcification causes some loss of cellular detail95 and can also affect the staining pattern^98,101. It may also be difficult to obtain good quality, thin (1-2 µm) sections of paraffin-embedded tissue. Alternatively, undecalcified tissue can be embedded in resin and thin (2 µm) sections prepared, to provide improved cellular morphology. One disadvantage of resin sections is the extended staining times required, but these can be significantly reduced when microwave-assisted techniques are used⁹⁷. These also produce more intense staining with improved contrast, however, the characteristic staining pattern is altered, with RNA-rich sites such as nucleoli and 'basophilic' cytoplasm staining purple rather than blue^97,102. A 24 hour protocol for processing and staining (H&E, Giemsa, reticulin) resin sections without microwaves has been described¹⁰³.

Lymphoid tissue can be processed routinely to paraffin wax or resin and thin (1-2 µm) sections prepared. Smears and imprints should be air-dried and fixed in methanol for 1 to 5 minutes before staining, depending upon the technique.

Factors affecting staining^{89,91,100,104}
THE STAIN
Stock stains
Stains are available commercially as dry powders or stock solutions. For storage, powders are most stable, and stock solutions in a methanol/glycerol solvent mixture are more stable than those in methanol alone. Different brands and batches can produce variable staining and therefore reagents should be carefully selected and the stain quality continually monitored¹⁰⁵. Stock stains should be stored in tightly capped polyethylene containers, in the dark at 4°C to prolong shelf life, as they are affected by moisture, contact with metals, exposure to light and variation in temperature.

Working solutions
Working stain solutions are prepared by diluting the stock with distilled water or an aqueous buffer. The buffer composition, and the concentration and pH of the working solution (6.0 to 8.5) all affect staining. The colour of erythrocytes, in particular, is altered by changes in the pH (pH 6.0 - red, pH 7.5 - colourless, pH 8.5 - bluish green)92. Working solutions are stable for only a few hours and should be freshly prepared in small volumes. Deterioration of dye solutions is evidenced by loss of red staining due to eosin precipitation¹⁰⁰.

THE STAINING PROTOCOL
The staining time must be long enough to achieve differential staining^97,106, but if excessive, then effective differentiation may not be possible⁹⁸. The best differential staining is achieved using a more dilute staining solution with a longer incubation time¹⁰¹. Differentiation is usually performed in two steps: 1) dilute acetic acid to remove excess blue staining and; 2) 95% ethanol to remove excess Eosin. These steps are difficult to standardise and sections must be processed individually under microscopical control.

Jenner Giemsa²⁸ for bone marrow sections
SPECIMEN PREPARATION
Cut 2 µm thick paraffin sections from tissue fixed in Zenker's fluid or 10% neutral buffered formalin. Bone marrow or tonsil are used as control tissue.

REAGENT PREPARATION
1 Jenner stock solution:
Jenner stain powder 1 g
Methanol 400 ml
(A commercial stock solution can be used).
Jenner working solution:
Jenner stock solution 25 ml
Distilled water 25 ml

2 Giemsa stock solution: Giemsa powder 1 g
Glycerol 66 ml
Methanol 66 ml
Mix the powder with the glycerol and place at 60°C for 2 hours. Add the methanol and mix well. (A commercial stock solution can be used).
Giemsa working solution:
Giemsa stock solution 2.5 ml
Distilled water 50 ml

3 1% acetic acid
Glacial acetic acid 0.5 ml
Distilled water 50 ml

METHOD
1 Dewax and rehydrate sections.
2 Remove mercuric pigment with iodine/thiosulphate if necessary.
3 Rinse in distilled water.
4 Rinse in two changes of methanol.
5 Place sections in working Jenner solution for 6 minutes.
6 Transfer to working Giemsa solution for 45 minutes.
7 Rinse rapidly in distilled water.
8 Differentiate in 1% acetic acid (control microscopically).
9 Rinse rapidly in distilled water.
10 Dehydrate rapidly, clear and mount.

TECHNICAL NOTE
May and Grünwald's solution can be used in place of Jenner's but requires a longer staining time (45 minutes).

Lennert's Giemsa⁹⁶ for lymph nodes
SPECIMEN PREPARATION
Cut 2 µm thick paraffin sections from tissue fixed in Zenker's fluid or 10% neutral buffered formalin. Lymph node or tonsil are used as control tissue.

REAGENT PREPARATION
1 Working Giemsa solution
Giemsa stock (Merck) 10 ml
Distilled water 40 ml

2 Acetic acid solution
Add 1-2 drops of glacial acetic acid to 50 ml distilled water.

METHOD
1 Dewax and rehydrate sections.
2 Remove mercuric pigment with iodine/thiosulphate if necessary.
3 Place sections in working Giemsa solution for 1 hour.
4 Rinse briefly in acetic acid solution.
5 Place in 96% ethanol until differentiated (control microscopically.
6 Rinse in two changes of isopropanol each of 2 minutes.
7 Clear and mount.

RESULTS (for both methods)
Nuclear chromatin - dark blue
Cytoplasm of lymphocytes and monocytes - pale blue
Neutrophil granules - purple
Eosinophil granules - pink/orange
Basophil granules - purple/black
Nucleoli - blue
Erythrocytes - pink
Connective tissue - pink to light purple
Mast cell granules - dark purple

Organelles
Suspended within the cytoplasm of eucaryotic cells are various structures¹⁰⁷ which are either: (a) membranous or membrane bound, perform specialised functions and form distinct subcompartments in their own right; or (b) are not membrane bound and, although they have specialised functions, do not form subcompartments within the cytoplasm. A variety of structures may be defined as organelles but here only those in group (a) are included. These are mitochondria, the endoplasmic reticulum, endosomes, peroxisomes, lysosomes, and the Golgi apparatus and its associated vesicles.

Organelles and other subcellular structures can only be visualised in a limited manner by conventional light microscopy. The smallest objects that can be seen are approximately 250 nm (0.25 µm) in diameter and, as most organelles are smaller than this, electron microscopy with its superior resolving power is more useful than light microscopical techniques which are restricted to mitochondria, the Golgi apparatus and lysosomes. However, histochemical and immunocytochemical techniques may also be used, with either light or electron microscopy.

Mitachondria
Mitochondria are approximately 0.5-1 µm in diameter and 2-5 µm in length and appear as threads or grains by light microscopy. All eucaryotic cells contain mitochondria that possess their own genome and are self replicating. Mitochondria contain a concentrated mixture of enzymes and are the major site of oxidative phosphorylation, supplying the cell's energy requirements by producing adenosine triphosphate (ATP).

PATHOLOGY
In normal cells mitochondria increase in number with increasing metabolic load and are also found in large numbers in oncocytomas. In the early stages of cellular injury both the mitochondria and endoplasmic reticulum may become swollen through an influx of water into the cell leading to cloudy swelling or hydropic change.¹⁰⁸ Structural alterations to mitochondria, such as pleomorphism, gigantism, ultrastructural abnormalities and inclusions, as well as quantitative changes, are found in a variety of neoplastic and non-neoplastic diseases11,¹⁰⁹ however, most of these changes cannot be demonstrated by light microscopy. In addition, enzyme alterations which occur in some disease states, are not always accompanied by morphological change109 but can be demonstrated histochemically.¹¹⁰

VITAL STAINS
In living tissue mitochondria can be visualised in unstained preparations by dark ground illumination or phase contrast microscopy. Alternatively, they can be demonstrated using supravital stains including: Janus Green B; Janus Blue; Janus Black; diethylsafranin; Pinacyanol; Rhodamine B; Methylene Blue;⁶⁶ and the fluorescent, cationic, lipophilic dye Rhodamine 123.¹¹¹ Mitochondria are sensitive to osmotic change and, in unfixed tissue, must be observed promptly and prior to autolysis.

JANUS GREEN B
Isolated cells are suspended in an isotonic solution containing Janus Green B (CI 11050) at a concentration of 1:10,000 (or weaker). Small portions of fresh tissue may be gently crushed in the same solution. Mitochondria stain a deep blue-green in approximately 5-10 minutes but the colouration is temporary. The methods for most other supravital stains are similar.

Pinacyanol-neutral red^112-113
Pinacyanol is claimed to be superior to Janus Green as it is less toxic and gives longer lasting preparations.

SPECIMEN PREPARATION
The technique may be used with blood (fresh, no anticoagulant), bone marrow, thin tissue slices, or crushed tissue fragments.

REAGENTS REQUIRED
1 0.4% aqueous Neutral Red (CI 50040) (a saturated solution)
2 0.1% Pinacyanol (CI (Ed.1) 808) in absolute alcohol
Mix 0.58 ml of the neutral red solution with 0.17 ml of pinacyanol. Make up to 5 ml with absolute alcohol. As an alternative, Lillie and Fullmer⁶⁶ recommend mixing 0.3 ml of pinacyanol with 1 ml of neutral red and then adding 8.7 ml of absolute alcohol to give a final volume of 10 ml.

METHOD
1 Coat clean slides with the stain mixture, drain and air dry.
2 Apply the specimen to a clean coverslip. The material is wetted with serum, or an alternative isotonic solution, and spread in a thin layer (preferably one cell thick) without air bubbles.
3 Place the coverslip onto a prepared stain-coated slide, sandwiching the specimen between the coated surface and the coverslip. Edges may be sealed with a suitable sealant (petroleum jelly).
4 Incubate for approximately 20 minutes at 37°C, or slightly longer at room temperature (monitor microscopically). Preparations made at room temperature last longer than those made at 37°C. It is recommended that observations are completed within 90 minutes, however, staining may remain for 2-4 days.

RESULT
Mitochondria - deep blue to violet, or purple in living cells
Nuclei - red

TECHNICAL NOTE
Pinacyanol is photosensitive, therefore both the coated slides and stock solution must be stored in the dark.¹¹² Coated slides last approximately two weeks and the stock solutions for up to 6 months.

Rhodamine 123¹¹¹
Rhodamine 123 is a specific vital fluorescent dye that stains mitochondria without first having to pass through endocytotic vesicles or lysosomes. High resolution staining is achieved with low background.¹¹¹ The dye has low cytotoxicity but dimethyl sulphoxide, recommended by some as an alternative solvent, is mildly cytotoxic and aqueous solutions are preferred.¹¹⁴

REAGENTS REQUIRED
1 Rhodamine 123
2 Dulbecco's modified Eagle's tissue culture medium
3 Double distilled water
Dissolve the rhodamine 123 in double distilled water at a concentration of 1 mg/ml, then dilute to 10 µg/ml in Dulbecco's modified Eagle's tissue culture medium.

METHOD
1 Incubate cultured cells (on coverslips) in the stain solution for 30 minutes in a 10% CO2 incubator at 37^oC.
2 Wash the cells in three changes of fresh medium without rhodamine each of 5 minutes.
3 Mount in fresh medium supplemented with 10% fetal calf serum.
4 Examine with a live cell observation chamber. Stained cells are viewed using epifluorescent illumination at either 546 nm (rhodamine excitation) or 485 nm (fluorescein excitation), or using confocal microscopy.

RESULTS
At 485 nm, mitochondria - green fluorescence, similar in colour to fluorescein excitation
At 546 nm, mitochondria - red fluorescence

FIXED TISSUE
A number of protocols based on the method of Altmann (1894)¹¹⁵ have been described for the demonstration of mitochondria in fixed tissue.^8,66 In this method the tissue is fixed in chrome-osmium, stained with a hot strong acid fuchsin solution and differentiated with picric acid.¹¹⁵ The methods described here are limited to those that are simple and easy to perform. As mitochondria are osmotically sensitive and quick to show post mortem change, small pieces of tissue should be fixed rapidly. To maximise resolution, material is embedded in ester wax and sections are cut at 2 µm, however, paraffin sections may also be used.

Phosphotungstic acid haematoxylin (PTAH)⁸
SPECIMEN PREPARATION
See Chapter 5.2
REAGENTS REQUIRED
1 PTAH (See Chapter 5.2)
2 4% aqueous iron alum

METHOD
1 Dewax and rehydrate sections.
2 Mordant in 4% iron alum for 20 to 60 minutes.
3 Wash well in distilled water.
4 Stain for 1-16 hours in PTAH as required. Alternatively sections can be stained in PTAH for 1-2 hours at 60°C.
5 Rinse very briefly in 95% alcohol.
6 Dehydrate, clear, and mount.

RESULTS
Nuclei and mitochondria - blue

Chromotrope-aniline blue (CAB)¹¹⁶
This stain is used for the demonstration of giant mitochondria, particularly in liver.
SPECIMEN PREPARATION
Cut 2 µm thick paraffin or 5 µm thick frozen sections from tissue fixed in 10% neutral buffered formalin. Fresh tissue can also be used for frozen sections.

REAGENTS REQUIRED
1 Chromotrope-aniline blue solution (CAB):
Hydrochloric acid (concentrated) 2.5 ml
Distilled water 200 ml
Aniline blue (CI 42780) 1.5 g
Chromotrope 2R (CI 16570) 6 g
Add the hydrochloric acid to distilled water then dissolve the aniline blue in the resulting solution, using gentle heat. Add chromotrope 2R and mix to dissolve. The final solution should have a pH of 1.0.
2 Weigert's iron haematoxylin
3 1% aqueous phosphomolybdic acid

METHOD
1 Dewax and rehydrate sections.
2 Stain nuclei with Weigert's haematoxylin.
3 Rinse in distilled water.
4 Immerse in 1% phosphomolybdic acid for 1-3 minutes.
5 Rinse in distilled water.
6 Stain with CAB solution for up to 8 minutes (ideally 2-4 minutes).
7 Rinse well in distilled water and blot dry.
8 Dehydrate quickly, clear, and mount.

RESULTS
Giant mitochondria - red or orange-red
Collagen - blue
Nuclei - black
TECHNICAL NOTE
In liver sections care should be taken not to confuse giant mitochondria with Mallory bodies that normally stain blue but may occasionally be red.

The Golgi apparatus
The Golgi apparatus was first described in animal neurones in 1898.^117-118 It is usually located near the nucleus and consists of a stack of flattened membrane bound cisternae that vary in number (usually from 3 to 12) and are approximately 1 µm in diameter.^107,109 Immediately adjacent are numerous small vesicles (~50 nm in diameter) which are thought to transport proteins and lipids from the endoplasmic reticulum to the Golgi and also between the cisternae. The cisternae are organised in a sequential series and material that enters the Golgi is modified and processed in a stepwise manner. Compounds are received into the primary cis compartment (forming face), pass to the medial cisternae (central), and then to the trans cisternae (maturing face). After sorting, material is distributed to the plasma membrane, lysosomes, and secretory vesicles.¹¹⁹ Histochemical techniques are useful for demonstrating Golgi, but only those performed at the ultrastructural level will reveal the contents (and thus the function) of the individual cisternae. In blood smears stained with H&E the Golgi apparatus can be seen in some cells (such as plasma cells) as a pale unstained area. In tissue sections the Golgi are demonstrated by impregnation with silver, osmium tetroxide, or both, however these techniques appear to stain only the cis face.¹²⁰ Special fixation is a prerequisite for these methods, they are also technically difficult and require experience to achieve good quality, consistent results. A number of methods (with variations) are described by Lillie and Fullmer.⁶⁶

Lysosomes
Lysosomes are a heterogenous group of membrane bound spherical or ovoid cytoplasmic vesicles ranging in size from 0.025 µm to 0.8 µm. They are defined biochemically by the presence of acid phosphatase but more than 60 lysosomal enzymes have been demonstrated, principally acid hydrolases with a small number of oxidoreductases121. Their main functions are summarised as follows:

• The digestion and recycling of cell organelles and phagocytosed material.
• Tissue reorganisation through the extracellular release of lysosomal enzymes.
• Tissue defence (the phagocytosis and destruction of infective organisms and the extracellular release of enzymes to attack parasites).
• Biochemical processing (the uptake and processing of compounds within lysosomes).
• Nucleocytoplasmic communication.

• There is active proliferation and AgNOR's are dispersed due to nucleolar dissociation.
• Nucleolar association is defective so that AgNOR's remain dispersed.
• Cell ploidy varies so that the number of AgNOR-bearing chromosomes either increases (polyploidy) or decreases (eg. monosomy).
• Transcriptional activity of ribosomal genes increases due to increased protein synthesis.

• The loss or addition of genetic material in chromosome re-arrangements.
• The origin of chromosomal aberrations.
• The origin of cells in culture.
• The clonal origin of tumours.
• The location of single genes.
• The zygosity of twins.
• Paternity.

• general background staining and non-specific silver deposition
• limited reliability and reproducibility
• staining of cytoplasmic granules in addition to NOR's
• fading of preparations during storage
• poor staining with some fixatives
• variability in the staining properties of individual NOR's and the effect that this has on their configuration and relative visibility.

• Specimens give the best results when freshly prepared.
• Metal equipment, including forceps, should be avoided.
• Solutions may be centrifuged before use.