Table 1 - Small biopsy (1 mm3 renal, liver and skin biopsies) ethanol/resin manual processing schedule (1 to 2 days)

Processing step	Time
Fixative (formalin or formol sublimate)	1 - 24 hours
50% Ethanol	15 minutes
70% Ethanol	15 minutes
95% Ethanol	15 minutes
95% Ethanol	30 minutes
Absolute Ethanol	30 minutes
Absolute Ethanol	30 minutes
Absolute Ethanol	60 minutes
Propylene Oxide	30 minutes
Propylene oxide/resin 50:50	60 minutes
Pure resin	60 minutes
Pure resin under vacuum (15 mm Hg)	60 minutes
Polymerise at 60°C	overnight
OR
Polymerise at 80°C	3 hours

Table 2 - Small biopsy (1 mm3 renal, liver and skin biopsies) acetone/resin manual processing schedule (1 day)

Processing step	Time
Fixative (formalin or formol sublimate)	2 - 24 hours
70% acetone	30 minutes
Absolute acetone	30 minutes
Absolute acetone	30 minutes
Absolute acetone	30 minutes
70% resin in acetone	60 minutes
Pure resin	90 minutes
Pure resin	90 minutes
Pure resin for polymerisation
Polymerise at 60°C	overnight

Table 3 - Small to medium biopsy (2 mm3 trephines, lymph node, spleen) acetone/resin manual processing schedule (2 days)

Processing step	Time
Fixative (formalin or formol sublimate)	2 - 24 hours
70% acetone	2 hours
Absolute acetone	90 minutes
Absolute acetone	90 minutes
Absolute acetone	2 hours
70% resin in acetone	overnight
Pure resin	3.5 hours
Pure resin	3.5 hours
Pure resin for polymerisation
Polymerise at 60°C	overnight

Table 4 - Large biopsy (3 mm3 cores of mineralised bone) acetone/resin manual processing schedule (3 days)

Processing step	Time
Fixative (formalin or formol sublimate)	48 hours
70% acetone	2 hours
Absolute acetone	90 minutes
Absolute acetone	90 minutes
Absolute acetone	2 hours
50:50 resin/acetone	overnight
70% resin in acetone	7 hours
90% resin in acetone	overnight
Pure resin	3 hours
Pure resin	3 hours
Pure resin for polymerisation	Vacuum embed at 60°C - 1 hour
Polymerise at 60°C	overnight

Table 5 - Large biopsy (3 - 5 mm3 large trephines, bone marrow, soft tissue biopsy) ethanol/resin manual processing schedule (3 days)

Processing step	Time
Fixative (formalin or formol sublimate)	overnight
70% ethanol	60 minutes
95% ethanol	60 minutes
95% ethanol	60 minutes
Absolute ethanol	60 minutes
Absolute ethanol	60 minutes
Absolute ethanol	60 minutes
Absolute ethanol	60 minutes
Absolute ethanol	overnight
Propylene oxide	30 minutes
Propylene oxide/resin 50:50	60 minutes
Propylene oxide/resin 25:75	120 minutes
Pure resin	overnight
Pure resin under vacuum (15 mm Hg)	4 hours
Polymerise at 60°C	overnight

Table 6 - Manual processing schedule (10 days) for large pieces of tissue processed using rotary mixer (10 mm2 x 2 mm undecalcified bone).

Processing step	Time
Fixative (formalin or formol sublimate)	up to 3 days
70% ethanol	12 hours
70% ethanol	12 hours
95% ethanol	12 hours
95% ethanol	12 hours
Absolute ethanol	12 hours
Absolute ethanol	12 hours
Absolute ethanol	12 hours
Absolute ethanol	12 hours
Acetone	12 hours
Acetone	12 hours
Acetone/resin 50:50	24 hours
Pure resin	24 hours
Pure resin under vacuum (15 mm Hg)	until bubbles stop appearing
Pure resin on rotator	24 hours
Pure resin under vacuum (15 mm Hg)	until bubbles stop appearing
Pure resin
Polymerise at 60°C	overnight
OR
Polymerise at 80°C	3 hours

Table 7 - Glycol methacrylate manual processing schedule for small biopsies. Polymerisation can be carried out with the block mould standing in cold water to help dissipate heat produced during polymerisation.

Processing step	Time
Fixative (formalin or formol sublimate)	2 to 3 hours
50% Ethanol	30 minutes
50% Ethanol	30 minutes
70% Ethanol	30 minutes
70% Ethanol	30 minutes
95% Ethanol	30 minutes
95% Ethanol	30 minutes
Absolute Ethanol	30 minutes
Absolute ethanol	30 minutes
Fresh GMA monomer at 4°C	8 hours
Fresh GMA monomer at 4°C	16 hours
Embed in fresh GMA polymer
Polymerise at 56°C in oxygen free atmosphere	8 hours minimum

Table 8 - Components of epoxy resins and resin mixtures¹²
EPOXY RESINS

Substance	Common Name or designation	Viscosity (mPa per second at 25°C)
1,4-butane diol diglycidyl ether	RD-2
Butyl glycidyl ether	DGE	23
1,2,7,8-diepoxyoctane	DEO	20.5
Diglycidyl ether of bis phenol A	Araldite, ACM, CY 212,	2500
Diglycidyl ether of polyethylene glycol	DER 736,
Propylene oxide
Triglycidyl ether of glycerol	Epon 812	1400
Vestopal W		900
Vinyl cyclohexane dioxide	VCD, ERL 4206	60

HARDENERS

Substance	Common Name or designation
Long chain acid anhydrides (a) dodecenylsuccinic anhydride (b) nonylsuccinic anhydride (c) n-hexylsuccinic anhydride	DDSA NSA HXSA
Methyl nadic anhydride	MNA

ACCELERATORS

Substance	Common Name or designation
Tris (dimethylamino methyl) phenol	DMP-30
Benzyl dimethyl amine	BDMA
Dimethyl amino ethanol	DMAE

Table 9 - Processing schedule for when enzymes are to be demonstrated. Dehydrants have been excluded.

Glycol methacrylate	Distilled water
25 parts	75 parts
50 parts	50 parts
75 parts	25 parts
80 parts	20 parts
90 parts	10 parts
95 parts	5 parts
100 parts	0 parts
100 parts	0 parts
Embed in polymerisation mixture at -30°C. Expose to UV light source until polymerisation is complete.

Table 10 - Recommended section thickness for a range of tissues and staining methods.

TISSUE	SECTION THICKNESS (µm)	STAINING METHOD*
Renal Biopsy	0.5 - 1	PASM
	1.5	H&E, PAS, PTAH, Trichrome
	2	MSB, Masson, Elastic Van Gieson
	4	Congo red
Lymph node, Spleen	1.5	H&E, Reticulin, Giemsa, PAS
Trephines	1.5	H&E, Perls’, Reticulin, Giemsa
Skin biopsy	1.5	H&E, PAS, Perls’, Trichrome
	2	Elastic Van Gieson, Masson, MSB
Liver biopsy	1.5	H&E, PAS, Perls’, Reticulin, Trichrome
Bone biopsy	1.5	H&E, Von Kossa-chlorazol blue
Neural tissue	3 - 4

* PAS (Periodic acid Schiff)
PASM (PAS - Methenamine silver)
PTAH (Phosphotungstic acid haematoxylin)
MSB (Martius yellow, brillliant crystal scarlet, aniline blue)