Immunohistochemistry (Paraffin)

*IMPORTANT: See the protocol on product webpage for the appropriate antibody dilution.

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: SignalStain^® Antibody Diluent (#8112)
7. 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of SignalStain^® EDTA Unmasking Solution (10X) (#14747) 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: SignalStain^® Boost IHC Detection Reagents (HRP, Rabbit #8114).
11. Substrate: SignalStain^® DAB Substrate Kit (#8059).
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

NOTE: This procedure describes the conditions that are recommended for the Biocare Medical Decloaking Chamber. Device-specific settings and operating instructions should be utilized for other pressure cookers.

1. Place slides in 250 ml room temperature EDTA unmasking solution in a 24-slide holder.
2. Place 500 ml dH₂O into the pressure cooker.
3. Place the slide holder into the pressure cooker, touching the heat shield. It may be advantageous to place a second 24-slide holder into the pressure cooker, filled with 250 ml water and blank slides.
4. Seal the chamber and proceed with retrieval. Settings for the Biocare Medical Decloaking Chamber follow.
   1. SP1 125°C 30 seconds
   2. SP2 90°C 10 seconds
5. Carefully vent the device, then remove the lid and cool the slides on the bench for 10 minutes.
6. Rinse the slides with dH₂O.

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 section 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 SignalStain^® Antibody Diluent #8112 to each section. Incubate overnight at 4°C.
7. Equilibrate SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
8. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
9. Cover section with 1-3 drops SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
10. Wash sections three times with wash buffer for 5 minutes each.
11. Add 1 drop (30 µl) SignalStain^® DAB Chromogen Concentrate to 1 ml SignalStain^® DAB Diluent and mix well before use.
12. Apply 100-400 µl SignalStain^® DAB to each section and monitor closely. 1-10 min generally provides an acceptable staining intensity.
13. Immerse slides in dH₂O.
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).