9. Immunohistochemistry for Parafinn-embedded tissue sections

Post date: May 26, 2018 5:03:08 PM

1. Place the slide on a hot plate (temperature 65^oC) for 3-4 minutes or till paraffin melts.

2. Dip the slides in a jar containing xylene for 10-15 min. Repeat this step in a 2^ndjar containing fresh xylene for 10 min.

3. Dip the slides in a jar containing 100% ethanol for 5 min. Repeat this step in a 2^ndjar containing fresh 100% ethanol for 5 min.

4. Dip the slides in a jar containing 90% ethanol for 5 min. Repeat this step in a 2^ndjar containing fresh 90% ethanol for 5 min.

5. Dip the slides in a jar containing 80% ethanol for 5 min. Repeat this step in a 2^ndjar containing fresh 80% ethanol for 5 min.

6. Dip the slides in a jar containing 70% ethanol for 5 min. Repeat this step in a 2^ndjar containing fresh 70% ethanol for 5 min.

7. Dip the slides in a jar containing dH2O for 5 min. Repeat this step in a 2^ndjar containing dH2O for 5 min.

8. Antigen Retrieval step- Put citrate buffer in a white container. Place the slides in a holder and then into the white container with buffer. Put saran wrap on it to avoid evaporation. [Antigen unmasking solution from Vector company:2.81 ml and 300 ml dH2O]

9. Microwave for 10 min. [Cook time: 10; Power level: 7] Press hold warm 3 times and hit start.

Alternatively, Microwave for 10 min. [Cook time: 10; Power level: 7]. Then cook time: 30 min and power level 0. Hit start. [For surface receptor detection, use less harsh treatment. DO not go above power level 7]. OR Place the slides in a holder and leave it in a pressure cooker or steamer. Check in between to make sure optimal pressure is reached. Once the pressure is reached, leave it for another 10-15 minutes. Then allow the pressure to be released. Take out the slides from pressure cooker and leave it at room temp. till it cools down.

10. Wash the slide in TBS for 5 min followed by 2 washes in dH2O for 5 min each wash.

11. Endogenous peroxidase quenching step: Incubate in 3% H2O2 (diluted in dH2O) in dark for 30 min.

12. Wash the slides with dH2O for 5 min.

13. Take out the slides from the container and lay horizontally in a slide box. Wipe off excess H2O from the slide without disturbing the tissue. Put a drop of dH2O in case the tissue starts drying up. Put a wide circle/square with Immedge pen to create a hydrophobic barrier.

14. Treat with 0.1% Triton-X-100 made in TBS for 5 min at room temp. This step will aid in permeabilizationfor intracellular staining. Skip this step for surface molecules.

15. Wash with TBS for 5 min. wipe off excess TBS.

16. Blocking step: Add 2% nonfat milk (milk is made in 0.1% Triton-X-100 in TBS) and incubate for 30 min at room temp.

17. Wash with TBS once for 5 min

18. Avidin-biotin blocking step:Some tissues may bind avidin, biotinylated horseradish peroxidase or other Biotin/Avidin System components without prior addition of biotinylated antibody. This binding may be due to endogenous biotin or biotin-binding proteins, lectins, or nonspecific binding substances present in the section. If a high background is present using the ABC reagents (or other avidin conjugate) in the absence of biotinylated secondary antibody, pre-treatment of the tissue with avidin, followed by biotin (to block the remaining biotin binding sites on the avidin), may be required. The blocking kit (supplied by vector technologies consists of an Avidin D solution and a biotin solution. Pre-treatment of the section with the Avidin D solution should always be followed by incubation with the biotin solution.

19. **Avidin-Biotin kit should be taken out 30 min prior to use. Add Avidin solution on top of the tissue (1 drop of avidin/ml of 2% nonfat milk (milk is made in 0.1% Triton-X-100 in TBS)).

20. Incubate for 15 min at room temp.

21. Wash 3 times with TBS [5 min each wash].

22. Add biotin solution on top of the tissue(1 drop of biotin/ml of 2% nonfat milk (made in 0.1% Triton-X-100 in TBS)).

23. Incubate for 15 min at room temp.

24. Wash 3 times with TBS [5 min each wash].

25. Primary Ab incubation step: Use appropriate dilution of 1^oAb [usually 1:50-1:100 dilutions is made in 2% nonfat milk (milk is made in 0.1% Triton-X-100 made in TBS)].

26. Incubate overnight at room temp. Create a moist environment inside the slide box by using wet paper towels.

27. Wash 3 times with 0.1% Triton-X-100 made in TBS [5 min each wash].

28. Secondary Ab incubation step: Add biotinylated 2^oAb [usually 1:200 dilutions is made in 2% nonfat milk (milk is made in 0.1% Triton-X-100 in TBS)].

29. Incubate for 1 h in dark at room temp.

30. Wash 2 times with 0.1% Triton-X-100 made in TBS [5 min each wash].

31. Add vectastain reagent from the vector company directly on the tissue. The R.T.U. VECTASTAIN® ABC Reagent allows more accessibility for binding to a biotinylated target and results in an increase in signal intensity with low background staining.

32. Incubate for 30 min at room temp.

33. Wash once with 0.1% Triton-X-100 made in TBS [5 min].

34. Add DAB solution [2.5 ml dH20 + 1 drop of buffer + 2 drops of DAB + 1 drop of H2O2 and mix].

35. Incubate at room temp. for few minutes till you see the brown color. ** DAB kit should be taken out at room temp. 15 min before use.

36. Wash with dH2O twice.

37. Counterstain with hematoxylin from vector company for few minutes till you see purple color.

38. Wash excess stain in dH2O.

39. Dehydrate the tissue by following steps.

40. Dip the slides in a jar containing 70% ethanol for 5-10 min. Repeat this step in a 2^ndjar containing fresh 70% ethanol for 5 min.

41. Dip the slides in a jar containing 80% ethanol for 5-10 min. Repeat this step in a 2^ndjar containing fresh 80% ethanol for 5 min.

42. Dip the slides in a jar containing 90% ethanol for 5-10 min. Repeat this step in a 2^ndjar containing fresh 90% ethanol for 5 min.

43. Dip the slides in a jar containing 100% ethanol for 5-10 min. Repeat this step in a 2^ndjar containing fresh 100% ethanol for 5 min.

44. Dip the slides in a jar containing xylene for 10-15 min. Repeat this step in a 2^ndjar containing fresh xylene for 10 min.

45. Allow few minutes for air drying and then put a drop of mounting solution on tissue and carefully place the coverslip. Allow a day or two for complete drying.