Histotechnology Technical methods

Immunohistochemical technique

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Fixation: 10% formol calcium.

Processing: Paraffin wax, with xylene, chloroform or Inhibisol as the clearing agent.

Sections: Between 3 and 10 microns in thickness. Mount on chromic acid etched and poly-L-lysine or chrome alum/gelatine coated slides. Dry at 37°C for at least 1 hour; overnight is preferable.


1. Dewax sections in xylene (5 mins. is adequate for 5 micron thick sections).

2. Rinse in a fresh bath of xylene.

3. Rinse in 2 baths of absolute alcohol.

4. Incubate in 300cm3. of absolute methanol containing 30cm3. 20 vol. hydrogen peroxide for 15 mins.

5. Rinse in 2 baths of absolute alcohol and then in running tap water. 5a. If trypsin digestion or another special pretreatment is required, insert the appropriate method here.

6. Incubate in 1:5 normal swine serum for 20 mins.

7. Drain, wipe off excess serum and incubate in primary antibody for 30 mins.

8. Jet wash in TRIS/HCl buffered saline, pH 7.6 (TBS).

9. Wash in TBS for 3 mins.

10. Incubate in biotinylated secondary antibody for 30 mins.

11. Jet wash in TBS.

12. Wash in TBS for 3 mins.

13. Incubate in Avidin/Biotin Complex (ABC) (see kit for instructions on how to make up) for 30 mins.

14. Jet wash in TBS.

15. Wash in TBS for 3 mins.

16. Incubate in DAB solution for 10 mins.

17. Wash in running tap water.

18. Incubate in 0.5% copper sulphate in 0.9% sodium chloride for 10 mins.

19. Wash in running tap water.

20. Counterstain in haematoxylin.

21. Dehydrate, clear . Mount sections in DPX


--Nuclei Blue

--Sites of peroxidase activity Black/Brown

DAB Solution

--Soln. A. 280cm3. TBS.

--Soln. B. 0.15g 3',3'diaminobenzidine tetrahydrochloride in 10cm3. TBS.

--Soln. C. 0.20g imidazole in 10cm3. TBS.

--Mix solns. A, B and C immediately prior to use.

--Incubate sections for 30 secs., add 225 microlitres of 20 vol. hydrogen peroxide and incubate for a further 10 mins.

0.005M TRIS/HCl buffered saline, pH 7.6 (TBS)

--NaCl 81g

--TRIS 6g

--1NHCl 42cm3.

--Distilled water 10ltrs.


1. Hydrogen peroxide is a powerful oxidizing agent; avoid skin contact.

2. DAB is a possible carcinogen; wear gloves when using it. The dry powder must never be used on the open bench; always use a fume cupboard.

3. It is possible that after use the copper sulphate solution in Step 18 may be contaminated with DAB; gloves should be worn when handling it.

4. Do not allow the sections to dry out at any stage during the technique.

5. Dilute primary and biotinylated secondary antisera as appropriate in 1:20 normal swine serum.

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.

© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk