Immunohistochemistry

1. Sections stored in PBS-azide should be rinsed with PBS 2x’s for 10 min. ea.  Sections stored in cryoprotectant should be rinsed with PBS 6x’s for 10 min. ea.

2. Incubate in 1% NaOH and 1% H₂O₂ for 20 min.  (300ml 30% NaOH and 300ml 30% H₂O₂ in 10ml ddH₂O)

3. Rinse in PBS 3x’s for 10 min. ea.

4. Incubate in 0.3% Glycine for 10 min.  (0.03gm glycine in 10ml PBS – IMPORTANT make fresh just before use!)

5. Rinse in PBS 3x’s for 10 min. ea.

6. Incubate in 0.03% SDS for 10 min.  (30ml 10% SDS in 10ml PBS)

7. Block in PBT-Azide w/ 3% Normal Serum [e.g. If Normal Serum was Donkey, the solution would be abbreviated PDT; for Normal Goat Serum, PGT]    (300ml Normal Serum in 10ml PBT).

8. Transfer sections into primary antibody and incubate at room temp. (or at 4^oC in cold room) on shaker ~50 rpm.  (primary antibody + 3% normal serum + PBT-Azide)            [anti-pSTAT3 antibody from Cell Signaling is diluted 1:4,000, incubate on shaker at rm. temp for 24 hours and then put in 4 ^oC for 24-48 hours]

9. Rinse with PBS 6x’s for 10 min. ea.

10. Transfer sections into secondary antibody for 1-2 hours on shaker ~50 rpm. [1:1000 for biotinylated-Jackson or -Vector]  (10ml of secondary antibody + 300ml normal serum + 10ml PBT) 

• Make ABC solution (1:500 = 10ml A  + 10ml B in 5ml PBS and allow to sit at RT for at least 30min. before use)

11. Rinse with PBS 3x’s for 10 min. ea.

12. Incubate in ABC* (Avidin Biotin Complex) for 1 hour.

13. Rinse with PBS 2x’s for 10 min. ea.

14. Incubate in 0.04% DAB and 0.01% H₂O₂ ~4-6 min.  (500ml DAB [+ 100ml 5% Cobalt Chloride + 

15. 500ml 1% Nickel Sulfate] + 50ml PBS; just before use add 16ml of 30% H₂O₂)

16. Rinse with PBS 2x’s for 10 min. ea.

17. Go on to next primary or store sections in PBS-Azide and/or mount onto gelatin coated slides.

* All steps done at room temperature on orbital shaker at a speed of ~30 rpm unless otherwise noted.

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