Abstract: Bone immunohistochemistry is a relatively tricky procedure. This protocol ensures that mouse spine tissue will stay on the slide throughout the process.

Procedure

Fixation/Embedding:

1. Harvest mouse spine.
2. Remove surrounding muscle tissue thoroughly and carefully.
3. Wash tissue in cold PBS then cut spine into 2 or 3 pieces (to later fit easily into a cassette)
4. Fix with 4% paraformaldehyde overnight at 4°C.
5. Transfer tissue into Decalcification Solution (Decalcifying Solution, Krajian, J.T.Baker, cat. # G161-02) for 4-5 days at 4°C.
6. Transfer tissue to a tissue cassette and store in 70% ethanol until tissue is processed and paraffin embedded.

Sectioning:

7. Cut bone sections at 7 micron thickness and place on to poly-L-Lysine coated slides.
8. Place slides on a slide warmer at 55°C for 90 minutes.
   
9. Store slides at room temperature overnight in an upright position.

De-waxing & Re-hydration:

10. Dunk slides in Citrosolv™ for 2 minutes (3X).
11. Dunk slides consecutively from 100%, 90%, 80%, and 70% ethanol for 2 minutes in each solution.
12. Re-hydrate tissue in distilled H₂O for 2 minutes (2X).

Antigen Retrieval:

13. Incubate tissue slides with working Proteinase K solution (Ref: http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm) in a humidified chamber for 7 minutes at 37°C.
14. Cool slides cool at room temperature for 5-7 minutes.
15. Wash in room temperature PBS for 2 minutes (2X).
16. Incubate slides in 0.2% Triton-X-100 solution in room temperature PBS for 15 minutes.
17. Wash slides in room temperature PBS for 2 minutes (2X).

Blocking:

18. Incubate tissue slides in 3:1 methanol containing 3% hydrogen peroxide for 30 minutes at room temperature.
19. Wash with room temperature PBS for 2 minutes (2X).
    
20. Incubate tissue in 10% animal serum for 1-2 hours (depending on antibody). Serum must not be from same animal as that from which antibody was prepared.
21. Wash tissue slides with room temperature PBS for 2 minutes.
    

Primary Antibody Incubation:

22. Incubate tissue with primary antibody at recommended dilution either for 1 hour at room temperature or overnight at 4°C.
23. Wash with room temperature PBS for 5 minutes (2X).

Secondary Antibody Incubation/Developing:

24. Prepare ABC reagents in the VectaStain Kit™ by first adding 2 drops of reagent A and then 2 drops of reagent B to 5 mls PBS.
25. Set solution at room temperature for 30 minutes.
26. Prepare secondary antibody at 1:1000 dilution in blocking serum which is prepared from the blue reagent in VectaStain Kit™.
27. Pipette solution containing the secondary antibody on to the tissue sections and incubate at room temperature for 30 minutes.
28. Wash slides with room temperature PBS for 2 minutes (2X).
    
29. Incubate tissue slides with ABC reagent (from Step 25) for 30 minutes.
    
30. Wash slides with room temperature PBS for 2 minutes (2X).
    
31. Prepare DAB working solution using the Peroxidase Substrate DAB Kit™ (Vector Laboratories) by mixing 2.5 ml distilled water, 1 drop of buffer solution, 2 drops of DAB, and 1 drop of peroxidase.
32. Stain tissues with DAB solution for approximately 1-2 minutes (or until brown stain develops, maximum 15 minutes).
    
33. Rinse tissue slides in distilled H2O for 1 minute (1X) and then 3 minutes (2X).
    
34. Stain tissues with 1:3 dilution of Harris modified Hematoxylin solution for 1-2 minutes.
    
35. Rinse tissue slides in distilled H₂O for 1 minute (1X), and 3 minutes (2X).
    

Dehydration & Mounting:

35. Dehydrate tissue slides consecutively in 70%, 80%, and 90% ethanol for 2 minutes in each solution at room temperature.
36. Set slides in 100% ethanol for 3-5 minutes.
    
37. Dunk tissue slides in Citrosolv™ for 2 minutes.
38. Allow to dry for about 30 seconds, and mount with VectaMount™ (Vector Laboratories).

Notes:
Protocol for Immunohistochemistry on Mouse Spine. Optimized by The Van Den Berg Lab, by Azadeh Nasrazadani. Last Modified: 8/2/07