Immunohistochemistry Protocol (Frozen)

A. Solutions and Reagents

1. Xylene
2. Ethanol (anhydrous denatured, histological grade 100% and 95%)
3. Hematoxylin (optional)
4. Fixative: For optimal fixative, please refer to the product data sheet
   1. 10% Neutral buffered formalin
   2. Acetone
   3. Methanol
   4. 16% formaldehyde
      1. 3% formaldehyde: To prepare, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
5. 10X Tris Buffered Saline (TBS): To Prepare 1 L add 24.2 g Trizma base (C₄H₁₁NO₃) and 80 g sodium chloride (NaCl) to 1 L dH₂O. Adjust pH to 7.6 with concentrated HCl.
6. Wash buffer: 1X Tris Buffered Saline (TBS) To prepare 1 L add 100 ml 10X TBS to 900 ml dH₂O.
7. Methanol/Peroxidase: To prepare, add 10 ml 30% H₂O₂ to 90 ml methanol. Store at -20°C.
8. Blocking Solution: 1X TBS/0.3% Triton-X 100/5% normal goat serum (#5425). To prepare: add 500 µl goat serum and 30 µl Triton-X 100 to 9.5 ml 1X TBS.
9. Biotinylated Secondary Antibody.
10. ABC Reagent: (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA). Prepare according to manufacturer’s instructions 30 minutes before use.
11. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

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

NOTE: Consult product data sheet to determine the optimal fixative.

1. After sections have dried on the slide, fix in optimal fixative as directed below.
   1. 10% Neutral buffered formalin: 10 minutes at room temperature. Proceed with staining procedure immediately.
   2. Cold acetone: 10 minutes at -20°C. Air dry. Proceed with staining procedure immediately.
   3. Methanol: 10 minutes at -20°C. Proceed with staining procedure immediately.
   4. 3% Formaldehyde: 15 minutes at room temperature. Proceed with staining procedure immediately.
   5. 3% Formaldehyde/methanol: 15 minutes at room temperature in 3% formaldehyde, followed by 5 minutes in methanol at -20°C (do not rinse in between). Proceed with staining procedure immediately.

D. Staining

1. Wash sections in wash buffer twice for 5 minutes.
2. Incubate for 10 minutes at room temperature in 3% H₂O₂ diluted in methanol.
3. Wash sections in wash buffer twice for 5 minutes.
4. Block each section with blocking solution for one hour at room temperature.
5. Remove blocking solution and add 100-400 µl diluted primary antibody to each section. (Dilute antibody in blocking solution). Incubate overnight at 4°C. *Refer to product datasheet to determine the recommended dilution.
6. Remove antibody solution and wash sections three times with wash buffer for 5 minutes each.
7. Add 100-400 µl secondary antibody, diluted in blocking solution per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
8. If using ABC avidin/biotin method, make ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
9. Remove secondary antibody solution and wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 µl ABC reagent to each section and incubate for 30 min. at room temperature.
11. Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
12. Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH₂O.
14. If desired, counterstain sections in Hematoxylin per manufacturer’s instructions.
15. Wash sections in dH₂O two times for 5 minutes each.
16. 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.
17. Mount coverslips.