Basic IHC Protocol - Frozen Sections on Slides

1. Remove slides from the freezer and allow to warm to room temp for 15min.
2. Using a PAP Pen, draw circles around sections.
3. Set slides in a staining box and apply 4% PFA to each slide. Incubate at room temp for 10 min. (This step helps to bind the section to the slide. Move to Step 5 if this is unnecessary!)
4. Rinse slides twice with PBS for 5min each.
5. Cover each section with 10mM Sodium Citrate buffer, pH 6. Process in the Biowave for 5min at 550W. (This step breaks some of the aldehyde-induced crosslinks, opening up epitopes for antigen binding. Move to Step 7 if antigen retrieval is unnecessary!)
6. Remove Citrate Buffer and replace with 1X PBS. Process slides for 1min each at 150W. Repeat.
7. Cover each section with 0.3% H2O2 (freshly made in dH2O). Process in the Biowave for 3min at 150W. (This step depletes endogenous peroxidases – do not skip!)
8. Remove H2O2 solution and replace with 1X PBS. Process slides for 1min each at 150W. Repeat.
9. Block sections in Carrier Solution for 1 Hr at RT.
10. Rinse slides briefly with PBS from a wash bottle.
11. Cover each section with 0.05% avidin solution from a Biotin-blocking kit. Incubate 15min at RT. (Avidin will bind endogenous biotin! Move to Step 15 if this is unnecessay!)
12. Rinse slides briefly with PBS from a wash bottle.
13. Cover each section with 0.005% biotin solution from a Biotin-blocking kit. Incubate 15min at RT.
14. Rinse slides briefly with PBS from a wash bottle, then apply PBS for 2x5min rinses.
15. Apply diluted primary antibodies to sections. Incubate at 4°C overnight.
16. Rinse each section briefly with PBS from a wash bottle, then transfer to a Coplin jar filled with PBS. Wash slides 5min, 15min, 5min with agitation. Use fresh PBS solution for each wash.
17. Apply diluted secondary biotinylated antibodies to sections. Incubate 1Hr at RT. **
18. Rinse each section briefly with PBS from a wash bottle, then transfer to a Coplin jar filled with PBS. Wash slides 5min, 15min, 5min with agitation. Use fresh PBS solution for each wash.
19. Apply prepared ABC Reagent to the sections. Incubate 30min at RT.
20. Rinse each section briefly with PBS from a wash bottle, then transfer to a Coplin jar filled with PBS. Wash slides 5min, 10min, 5min with agitation. Use fresh PBS solution for each wash.
21. Working with only a few slides at a time, apply freshly prepared DAB solution to sections. Set a timer to count up and allow brown color to develop. (Generally 2 – 10min)
22. Rinse slides briefly with dH2O and place in a Coplin jar of dH2O until all slides are developed.
23. Mount coverslips using MOWIOL.

Carrier Solution: Makes 10ml (enough to process 10 slides)

8.0ml of dH2O

1.0ml 10X PBS, pH 7.4

0.15ml 20% Triton (0.3% Final)

0.05g BSA (0.5% Final)

0.10ml Normal Serum (1% Final; must match host of secondary antibody – can be found in the Vectastain ABC Kit.)

Bring to 10ml final volume with dH2O.

Check pH and adjust as necessary to 7.4.

Primary Antibody: Dilute in Carrier Solution to desired concentration.

Secondary Antibody: Dilute Biotinylated Antibody from ABC Kit in Carrier

Solution to desired concentration.

**Prepare the ABC solution 30min into the secondary incubation. This solution must sit for at least 30min prior to use, but can be kept for hours at room temp.

Vectastain Elite ABC Kit:

Add 2 drops of Reagent A to 5ml of PBS and mix well. Add 2 drops of Reagent B and mix again.

DAB Substrate Kit: Just before use:

Add 2 drops of Buffer Stock to 5ml dH2O; mix well.

Add 4 drops of DAB Stock Solution; mix well.

Add 2 drops of H2O2 Solution; mix well.

If a gray-black color is desired,

Add 2 drops of Nickel Solution; mix well.