Immunohistochemistry (Frozen)

A. Solutions and Reagents

1. Xylene
2. Ethanol (anhydrous denatured, histological grade 100% and 95%)
3. Deionized water (dH₂O).
4. Fixative: 10% neutral buffered formalin.

5. Wash Buffer: 1X Tris Buffered Saline (TBS).

   To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH₂O, mix.

6. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
7. Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
   1. 1X TBS/0.3% Triton X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton X-100 to 9.5 ml 1X TBS.
   2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
8. Antibody Diluent 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
9. Detection System: VECTASTAIN^® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
10. Substrate: Vector^® NovaRED™ (Vector Laboratories).
11. Hematoxylin: Hematoxylin (#14166).
12. Mounting Medium: SignalStain^® Mounting Medium (#84583).

B. Sectioning

1. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. This may prevent cracking of the block when sectioning.
2. Section tissue at a range of 6-8 µm and place on positively charged slides.
3. Allow sections to air dry on bench for a few minutes before fixing (this helps sections adhere to slides).

C. Fixation

1. Fix sections in 10% NBF for 10 min at room temperature. Proceed with staining procedure immediately (Section D).

D. Staining

1. Wash sections in wash buffer twice for 5 minutes.
2. Incubate for 10 minutes at room temperature in 3% Hydrogen Peroxide.
3. Wash sections in wash buffer twice for 5 minutes.
4. Block each section with 100-400 µl of preferred blocking solution for one hour at room temperature.
5. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section.
6. Incubate overnight at 4°C.
7. Prepare ABC solution per manufacturer's recommendations.
8. Add 100-400 µl biotinylated secondary antibody, diluted in wash buffer per manufacturer's recommendation, to each section. Incubate 30 minutes at room temperature.
9. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
10. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
11. Wash section three times with wash buffer for 5 min each.
12. Prepare Vector® NovaRED per manufacturer's recommendations.
13. Apply 100-400 µl substrate to each section and monitor closely. 1-10 min generally provides an acceptable staining intensity.
14. Immerse slides in dH₂O.
15. If desired, counterstain sections in hematoxylin (#14166).
16. Wash sections in dH2O two times for 5 min each.
17. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
18. Mount sections with coverslips and mounting medium (#84583).