Histotechnology Technical methods

Immunohistochemical Stain

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Preparation: Cytological Preparations

Fixation: Air dry films or cytospin preparations overnight at room temperature. For frozen and paraffin sections refer to the respective 'indirect' techniques.


1. Fix cytological preparations in acetone at 4C. 30 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.

Steps 7-9 for POLYCLONAL Primary Antisera Only

7. Incubate in mouse anti-rabbit immunoglobulins. 30 minutes.

8. Jet wash in buffer.

9. Wash in buffer. 3 minutes.

10. Incubate in rabbit anti-mouse immunoglobins. 30 minutes.

11. Jet wash in buffer.

12. Wash in buffer. 3 minutes.

13. Incubate in alkaline phosphatase-mouse anti-alkaline phosphatase 30 minutes.

14. Jet wash in buffer.

15. Wash in buffer. 3 minutes.

16. Incubate in rabbit anti-mouse immunoglobins. 10 minutes.

17. Jet wash in buffer.

18. Wash in buffer. 3 minutes.

19. Incubate in alkaline phosphatase-mouse anti-alkaline phosphatase. 10 minutes.

20. Jet wash in buffer.

21. Wash in buffer. 3 minutes.

22. Incubate in substrate solution. 15 minutes.

23. Jet wash in buffer.

24. Wash in tap water.

25. Counterstain nuclei in haematoxylin LIGHTLY. DO NOT DIFFERENTIATE IN ACID ALCOHOL.

26. Mount in an AQUEOUS MOUNTANT.


--Nuclei Blue

--Sites of alkaline phosphatase activity Red

Substrate Solution

Naphthol-AS-MX phosphatase, free acid (Sigma N 4875) 2 mg

N,N-Dimethyl formamide 0.2 ml

0.1M Tris buffer pH 8.2 9.8 ml

Levamisole (Sigma L 9756) 2.4 mg

Fast Red TR salt (Sigma F 1500) 10 mg

Dissolve naphthol-AS-MX phosphatase, free acid in N,N-dimethyl formamide. Add the Tris buffer. Dissolve and Fast Red TR in the naphthol-Tris buffer and filter immediately before use.

0.005M Tris/HC1 Saline Buffer pH 7.6

--NaCl 81 g

--Tris 6 g

--NHCl 42 mls

--Distilled water 10 Litres

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