You will need the following reagents:-

Antibodies Normal Swine Serum (NSS), 1:5 in Tris buffer. 1°: antibody, diluted in 1/20 NSS/Tris. 2° antibody, diluted in 1/20 NSS/Tris.

Streptavidin/biotin complex StreptABComplex HRP (Dako K377). Prepare as per instructions.

DAB and Imidazole

• A) 0.15g 3,3''diaminobenzidine tetrahydrochloride in 10ml Tris buffer. B) 0.20g imidazole in 10ml Tris buffer. Add solutions A and B to 280ml Tris buffer, incubate slides in this solution for 30 seconds prior to the addition of 45ul of hydrogen peroxide, incubate for 10 minutes. N.B.: Solutions A and B must be made up immediately prior to use, or stored frozen and thawed out in the dark.

0.05M Tris/HCl Saline Buffer pH 7.6

Add to 10 litres of distilled H2O, 81g NaCl, 6g Tris and 42ml conc. HCl. Adjust pH to 7.6.

Scot''s Tap Water Substitute

Add to 2 litres of tap water, 4g potassium bicarbonate and 40g MgSO4.

Acid Alcohol

1ml concentrated HCl in 99ml 70 industrial methanol.

0.5 CuSO4 in 0.9 NaCl

Add to 1 litre of distilled H2O, 5g CuSO4.5H2O and 9g NaCl.

METHOD

1) Dewax sections for 5 minutes in xylene.

2) Rinse in fresh xylene.

3) Rinse twice in ethanol.

4) Incubate in 392ml absolute methanol plus 8ml 100 vol hydrogen peroxide for 15 minutes.

5) Rinse twice in absolute ethanol, and then in running tap water.

6) Incubate in 20 normal swine serum for 20 minutes.

7) Drain off excess serum onto post lip paper, wipe slide as dry as possible with tissue.

8) Incubate with primary antibody for 30 minutes in a humid chamber.

9) Jet wash with Tris buffer, followed by a 3 minute wash in a Tris bath.

10) Incubate in biotinylated secondary antibody for 30 minutes in a humid chamber.

11) Jet wash in Tris buffer.

12) Wash in Tris bath for 3 minutes.

13) Incubate in Avidin/Biotin complex, (which has to be prepared at least 30 minutes before use), for 30 minutes..

14) Jet wash in Tris buffer.

15) Wash in Tris bath for 3 minutes.

16) Incubate in DAB solution for 10 minutes.

17) Wash in running tap water.

18) Incubate in copper sulphate in 0.9 sodium chloride for 10 minutes.

19) Wash in running tap water.

20) Counter stain in haemotoxylin:-

• a) 1 minute in Harris''s haemotoxylin. b) Wash in tap water. c) 1/2 minute in Scot''s tap water substitute. d) Wash in tap water. e) Differentiate in acid/alcohol. f) Wash in tap water. g) 1/2 minute in Scot''s tap water substitute. h) Wash in tap water.

21) Dehydrate slides:-

• a) 100 alcohol until streaks stop b) " " " " " c) " " " " " d) 100 xylene " " " e) " " " " " f) " " " " "

22) Mount slides in DPX whilst still wet.

NOTES

1) Do not let slides dry out during this process. 2) Hydrogen proxide is a powerful oxidising agent, avoid skin contact. 3) DAB is a possible carcinogen.