INDIRECT PEROXIDASE TECHNIQUE

Fixation: Fresh tissue

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Sections: Thickness-Between 5 and 10 microns. Pick up on chromic acid etched, poly-L-lysine or chrome alum/gelatine coated slides. Dry at room temperature overnight (not longer than 72 hours).

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Technique

1. Fix sections in acetone at room temperature. 20 minutes.

2. Transfer as quickly as possible to a drying cabinet set a room temperature. 15 minutes.

3. Incubate in 1:5 normal swine serum. 20 minutes.

4. Drain and wipe off excess serum and incubate in primary antibody. 30 minutes.

5. Jet wash in tris/HC1 saline buffer pH 7.6.

6. Wash in buffer. 3 minutes.

7. Incubate in secondary antibody. 30 minutes.

8. Jet wash in buffer.

9. Wash in buffer. 3 minutes.

10. Incubate in DAB solution. 10 minutes.

11. Wash in running tap water.

12. Incubate in 0.5% CuS04.5H20 in 0.9% NaC1. 10 minutes.

13. Wash in running tap water.

14. Counterstain in haematoxylin.

15. Dehydrate, clear . Mount sections in DPX

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Results

--Nuclei Blue

--Sites of peroxidase activity Black/brown

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DAB Solution

--SOLN. A 280 mls tris/HC1 saline buffer

--SOLN. B 0.15 g 3,3 diaminobenzidine tetrahydrochloride in 10 mls buffer.

--SOLN. C 0.20 g imidazole in 10 mls buffer.

--SOLN. D Mix solns. A, B and C.

Incubate in soln. D for 30 seconds, add 225 microlitres of 20 volume hydrogen peroxide, and incubate for a further 10 minutes.

0.005M Tris/HC1 Saline Buffer pH 7.6

--NaC1 81 g

--Tris 6 g

--NHC1 42mls

--Distilled water 10 Litres

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Notes

1. Hydrogen peroxide is a strong 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 the copper sulphate at step 12 may be contaminated with DAB. Gloves should therefore be worn when using the copper sulphate solution.

4. Avoid dropping any of the reagents directly onto the sections.

5. Do not allow sections to dry out during the technique.

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.

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© 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