Immunohistochemistry (Paraffin)

A. Solutions and Reagents

1. Xylene
2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
3. Deionized water (dH₂O)
4. Hematoxylin (optional)
5. Wash Buffer:
   1. 1X Tris Buffered Saline with Tween^® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween^® 20 (#9997) to 900 ml dH₂0, mix.
6. Antibody Diluent PBST/5% normal goat serum (#5425): To 5 ml 1X PBST, add 250 µl Normal Goat Serum (#5425).
   1. 1X PBST: To prepare 1X PBST, add 50 ml Phosphate Buffered Saline with Tween^® 20 (PBST-20X) (#9809) to 950 ml dH₂O, mix.
7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
   1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
   2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
10. Detection System: VECTASTAIN^® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
11. Substrate: Vector^® NovaRED™ (Vector Laboratories).
12. Hematoxylin: Hematoxylin (#14166).
13. Mounting Medium: SignalStain^® Mounting Medium (#84583).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:
   1. Incubate sections in three washes of xylene for 5 minutes each.
   2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
   3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
2. Wash sections twice in dH₂O for 5 minutes each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

1. Wash sections in dH₂O three times for 5 minutes each.
2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
3. Wash sections in dH₂O twice for 5 minutes each.
4. Wash sections in wash buffer for 5 minutes.
5. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
6. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
7. Prepare ABC solution per manufacturer's recommendations.
8. Add 100-400 µl biotinylated secondary antibody, diluted in TBST 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 tempature.
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 minutes generally provides an acceptable staining intensity.
14. If desired, counterstain sections with hematoxylin (#14166).
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 sections with coverslips and mounting medium (#84583).