Mountants with a refractive index (RI) as close as possible to that of the tissue (usually taken as the RI of fixed protein, between 1.53 and 1.54) will effectively render a section transparent. With such specimens the only features visible will be those coloured by the staining method used. A medium with a much lower or higher RI than that of the tissue will increase visibility but is beneficial only if an overview of the specimen is required as the resolution can be poor (evident in the extreme if sections are cleared and examined dry; the mountant in this case, air, has a RI of 1.000). In some forms of microscopy (such as phase contrast) the use of a mountant with an RI only slightly different from that of tissue is recommended to supplement contrast enhancement.

To be effective, a mountant should possess certain characteristics. These include the following^1-2:

• it should be colourless and transparent
• it should be able to completely permeate and fill tissue interstices
• it should have no adverse effect on tissue components
• it should be resistant to contamination (particularly microorganism growth)
• it should protect the section from physical damage and chemical activity (oxidation and changes in pH)
• it should be completely miscible with dehydrant or clearing agent
• it should set without crystallising, cracking or shrinking (or otherwise deform the material being mounted) and not react with, leach or induce fading³ in stains and reaction products (including those from enzyme histochemical, hybridisation and immunohistochemical procedures)
• finally, once set, the mountant should remain stable (in terms of the features listed above).

Types of mountants
Mounting media are either hydrophobic or hydrophilic. Methods using the former generally call for sections to be dehydrated (usually through a series of graded ethanol solutions) and cleared (in a solvent miscible with the mountant) before the mounting medium is applied. Sections are mounted in hydrophilic media directly from water.

Within each group, mountants can also be classed as adhesives or non-adhesives1. In general, adhesives harden through solvent evaporation and thereby fix the accompanying coverslip to the slide. During this process the RI of the medium alters, moving away from that of the solvent and towards that of the dry mountant.⁴ The exact RI of the applied medium cannot therefore be known.⁵ Nevertheless as the RI of hydrophobic (adhesive) mountants usually approximates that of tissue proteins (fixed), and they provide firm adhesion of the coverslip, these mountants are the type most frequently used.

Hydrophilic media, although of relatively low RI, are essential for procedures in which dehydrants and hydrocarbon type clearants must be avoided. This will mostly relate to methods for demonstrating lipid, enzyme identification and immunohistochemistry. Disadvantages of hydrophilic mountants are that they may induce stains to leach from the section and many, being non adhesive, remain soft such that the edge of the coverslip must be sealed to prevent drying out. Suitable agents (ringing media) for this purpose are nail varnish, resinous mountants (see later) or paraffin wax.⁶

Hydrophobic mountants
CANADA BALSAM
This is an oleoresin obtained from the bark of the fir Abies balsamea (of the family Pinaceae), native to North America. The dried resin is freely soluble in xylene and other organic solvents. Originally introduced in about 1832 and widely used until only recently, Canada balsam has a number of disadvantages: it yellows with age; is very slow to harden and; as it becomes increasingly acidic over time, cationic dyes are poorly preserved and the Prussian blue product of Perls' reaction is bleached.^5,7

REAGENTS REQUIRED
1 Canada balsam 55-65 g
2 Xylene 100 ml

METHOD
Prepare using the quantities indicated (greater amounts of resin will result in more viscous solutions). RI (solution) 1.523.

DPX (DISTRENE, PLASTICISER, XYLENE)^8-9
One of the most commonly used mountants, DPX is a colourless, neutral medium in which most standard stains are well preserved. It is prepared by dissolving the common plastic, polystyrene, in a suitable hydrocarbon solvent (usually xylene). A major disadvantage of polystyrene media, however is that they set quickly and in doing so often retract from the edge of the coverslip. This can be prevented by adding a plasticiser which is thought to resist the effect by forming a mesh with the polymerised plastic.^1,10

REAGENTS REQUIRED
1 Polystyrene (Distrene 80) 18 g
2 Dibutyl phthalate 7.5 ml
3 Xylene 52.5 ml

METHOD
Prepare using the quantities indicated. RI (solution) 1.523.

EUPARAL⁵
Euparal is a mixture of eucalyptol, sandarac (a resin from the tree, Tetraclinin articulata grown in north west Africa), paraldehyde and camsal (camphor and phenyl salicylate). Its relatively low RI (which is usually given as 1.483 but ranges from 1.478 at 20°C to 1.535 when solid) makes it useful for mounting unstained sections. Another advantage is that slides may be transferred directly from 95% alcohol eliminating the need for complete dehydration and clearing. Some fading may occur in haematoxylin stained sections; in this situation the green (or 'vert') copper-containing form of Euparal is advocated.⁴

RESIN-EMBEDDED TISSUE
Sections of tissue embedded in plastic compounds (such as epoxy resins) can be successfully mounted in liquid resin of the same type. Sections should be completely dry before applying mountant which is best set using the same conditions prescribed for tissue blocks.

PHOTOSENSITIVE RESINS
Light polymerising resins have the advantage of very short setting times, requiring in the order of 10-30 seconds exposure to UV light to harden completely. Once cured, however, the mountant cannot be dissolved nor the coverslip removed^11-12 (as might be necessary for restaining). Acrylic based light sensitive resins are also suitable for fluorescence microscopy.

Hhydrophilic (aqueous) mountants
WATER
Although of low RI (1.333), water serves as a convenient temporary mountant for some whole specimens¹³ for examining certain microorganisms live (saline mount) and particularly when checking sections during staining procedures.

GLYCEROL
Glycerol is also a useful temporary mountant but with a higher RI (1.460) and longer drying time than water. Ringing the coverslip with a hydrophobic seal will extend the life of mounted sections, although cationic dyes will diffuse into the medium over time. Phosphate buffered glycerol (RI 1.47) is commonly used to mount sections for immunofluorescence¹⁴ and glycerol may be added to other agents to retard drying and cracking5.

REAGENTS REQUIRED
1 0.2 mol/l phosphate buffer 1 part
2 Glycerol 9 parts

METHOD
Prepare using the quantities indicated.

The addition of gum arabic (derived from species of Acacia) to a solution of glycerol will result in an adhesive mountant (Farrant's medium) with a RI of 1.436. The preparation is acidic but rendered neutral through the inclusion of potassium acetate6 which also retards dye extraction from methyl violet stained sections of amyloid.⁵

REAGENTS REQUIRED
1 Gum arabic (acacia gum) 50 g
2 Glycerol 50 ml
3 Distilled water 50 ml
4 Arsenic trioxide 1 g
5 Potassium acetate 50 g

METHOD
Dissolve gum arabic in warmed water then add other ingredients.

TECHNICAL NOTE
It is usual to incorporate arsenic as a preservative but thymol (100 mg), merthiolate (15 mg) or cresol (0.1 ml) are effective substitutes.

GLYCERINE (GLYCEROL) JELLY
This commonly utilised aqueous mountant is a mixture of glycerol and gelatine and has a RI of 1.47. It should set quite hard but for long term preservation sections are best ringed and sealed. Various formulations are in use.^16-19

APATHY'S MEDIUM²⁰
Sucrose added to gum arabic preparations increases the RI and prevents overdrying. The inclusion of potassium acetate will prevent leaching of metachromatic dyes.²¹

REAGENTS REQUIRED
1 Gum arabic 20 g
2 Cane sugar 20 g
3 Potassium acetate 20 g
4 Distilled water 40 ml
5 Thymol 0.02 g

METHOD
Dissolve components in warmed distilled water. Store in air tight containers.

POLYVINYL ALCOHOL
Polyvinyl alcohol, often used as a mountant in immunofluorescence microscopy, has been recommended as an alternative for glycerine jelly.²² Adding paraphenylenediamine to the preparation is effective in retarding photo fading.^23-24

Mounting sections
Mounting a suitably prepared specimen for microscopy is one of the most common laboratory procedures encountered. With a little practice, determining the type and quantity of mountant required and affixing a coverslip can be achieved without difficulty.

The method of storing and dispensing a mountant will depend largely upon the characteristics of the solvent in which the mountant is prepared. Although glass containers and droppers (or a glass rod dipped into the mountant) are universally suitable they are not practical for media with highly volatile, organic solvents (such as resinous agents). Continually opening the jar encourages solvent loss through evaporation leading to increased viscosity in the mountant. Solvent-resistant plastic syringes are an effective delivery system except that needles (if used) must be flushed regularly otherwise any mountant therein will set. A more useful approach is to use metal (tin) tubes. These are filled at one end which is then folded over and pinched tight to prevent leakage. Mountant is dispensed through a fine nozzle at the opposite end which is covered (preferably with a screw cap) when not in use.

METHOD
1 Select the appropriately sized coverslip and place on a white paper sheet.
2 Place a drop of mountant in the centre of the coverslip.
3 Remove surplus clearant from the back of the slide and around the section, leaving a margin of approximately 3 mm. The section should not be allowed to dry out (unless this is specified in the procedure being undertaken).
4 Invert the slide (section face down) over the coverslip and with one end resting on the paper sheet, gradually lower the other end until the mountant touches the section. Mountant will spread quickly over the section, between slide and coverslip. The slide, with coverslip attached, is then turned upright. Any trapped air is gently squeezed out whilst aligning the coverslip.
5 The mountant is allowed to set. The time required will depend upon the particular agent used but in some cases warming the slides (37°-60°C) will hasten the process. If the result is inadequate, slides are returned to the solvent (appropriate to the mountant) to have the coverslip removed and the process repeated.

Restaining
In some instances it becomes necessary to gain access to a section previously set in mountant: to restain specimens that have faded appreciably, remove the original stain and apply a new technique or to superimpose an additional stain. Mounted tissue may also need to be retrieved for additional assessment such as by electron microscopy or X-ray analysis.

In this case, coverslips can be removed by soaking in the solvent appropriate to the mountant. This may require one to two days after which the coverslip is gently removed and sections are rehydrated and treated as required. Alternatively, slides can be left at -20°C for up to one hour then a sharp blade used to carefully prise off the coverslip.

Labelling and storage of slides
Slide mounted sections are identified during preparation by inscribing the slide with the tissue accession number or suitable code using a diamond marker or pencil (frosted slides). Care should be exercised when using characters such as A, I, O, T, V and the like as these appear the same when viewed from either side of the slide; incorrectly identifying the side upon which the section is mounted may lead to poor staining or section damage.

After staining and mounting it is common practice to affix to the slide a paper label upon which the laboratory number, staining method and date are usually written. This may be supplemented with a bar code or other symbol which can be recognised by a computer supported image scanner.

It is important when attaching identifying labels to be aware of the slide storage system to be utilised. Standard 7.5 x 2.5 (3" x 1") slides are usually stored, standing on their short side, in metal or plastic drawers. Labels need to be fixed to the opposite end and orientated so the details can be read with the slide in this upright position.

Coverslips
To be compatible with the standard slide, coverslips are 22 mm or 24 mm wide but vary in length to suit the size of the section. In general a selection of 22 mm, 30 mm, 50 mm and possibly 60 mm (if preparing cell smears) will accommodate most purposes. Round coverslips of different diameters are also available.

Coverslips are manufactured to a specified thickness to complement the optical specifications of microscope objectives lenses (the recommended thickness is indicated on the barrel of the lens and is normally 0.17 mm). Various thicknesses are available (with some variation between manufacturers) and are designated No. 1 (0.13 - 0.17 mm), No. 1½ (0.16 - 0.19 mm) and No. 2 (0.19 - 0.25 mm).

References
1 Baker JR. Cytological Technique. 5th ed. London: Science Paperbacks. 1966

2 Lynch MJ, Raphael SS, Mellor LD, Spare PD, Inwood MJH. Medical laboratory technology and clinical pathology. 2nd ed. Philadelphia: WB Saunders. 1969

3 Barr WT. Effects of sunlight on stained sections mounted in various media. Stain Technol 1970; 45: 9-14

4 Lillie RD, Fullmer HM. Histopathologic technic and practical histochemistry. 4th ed. New York: McGraw-Hill. 1976

5 Lillie RD, Zirkle C, Dempsey EW, Greco JP. Final report of the committee on histologic mounting media. Stain Technol 1953; 28: 57-80

6 Culling CFA. Handbook of histopathological and histochemical techniques. 3rd ed. London: Butterworths. 1974

7 Lillie RD, Windle WF, Zirkle C. Interim report of the committee on histologic mounting media: resinous media. Stain Technol 1950; 25: 1-9

8 Kirkpatrick J, Lendrum AC. Mounting medium for microscopical preparations giving good preservation of colour. J Path Bact 1939; 49: 592-594

9 Kirkpatrick J, Lendrum AC. Further observations on the use of synthetic resin as a substitute for Canada balsam; precipitation of paraffin wax in medium and an improved plasticiser. J Path Bact 1941; 53: 441-443

10 Lyon H. Tissue processing: VII. Post treatment. In Lyon H ed. Theory and strategy in histochemistry. Berlin: Springer-Verlag. 1991

11 Denton J. Loctite UV 357 polymerised glass adhesive as a section mounting medium. Technical Methods 1986; 40: 584-585

12 Silverman M. Light-polymerising plastics as slide mounting media. Stain Technol 1986; 61: 135-137

13 Stankler L. The use of water as a mounting medium for light microscopy to study the surface architecture of hair. Br J Dermatol 1989; 120: 719-721

14 Coons AH, Creech HJ, Jones RN, Berliner E. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 1942; 45: 159-170

15 Kaiser E. Instrumente, Präparirungs- u. Conservirungsmethoden etc. Verfahren zur Herstellung einer tadellosen Glycerin-Gelatine. Bot Centralb 1880; 1: 25-26

16 Weeks RJ, Padhye AA. A mounting medium for permanent preparations of microfungi. Mykosen 1982; 25: 702-704

17 Bancroft JD, Stevens A Eds. Theory and practice of histological techniques. 3rd ed. Edinburgh: Churchill Livingstone. 1991

18 Pearse AGE. Histochemistry. Theoretical and applied. 3rd ed. Edinburgh: Churchill Livingstone. 1972

19 Mallory FB. Pathological technique. Philadelphia: Saunders. 1938

20 Apathy, S. Erfahrungen in der Behandlung des Nervensystems für histologische Zwecke. Z wiss Mikr 1892-1893; 41: 559-562

21 Highman B. Improved methods for demonstrating amyloid in paraffin sections. Arch Path 1946; 41: 559-562

22 Zaman G, Chayen J. An aqueous mounting medium. J Clin Pathol 1981; 34: 567-568

23 Balatan AJ, Dalix AM, Oriol R. An improved mounting medium for immunofluorescence microscopy. Arch Pathol Lab Med; 109: 108

24 Ju G, Han Z, Fan L. Fluorogold as a retrograde tracer in combination with immunohistochemistry. J Neurosci Methods 1989; 29: 69-72