ALKALINE PHOSPHATASE (APAAP) TECHNIQUE

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.

Technique

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.

Results

--Nuclei Blue

--Sites of alkaline phosphatase activity Red

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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