Immunohistochemistry Protocol (Paraffin) - Specific For Products: 2085, 3197

*IMPORTANT: See product data sheets 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:
   1X TBS/0.1% Tween-20 (1X TBST): To prepare 1 L add 100 ml 10X TBS to 900 ml dH₂O. Add 1 ml Tween-20 and mix.
   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. Antibody Diluent: SignalStain^® Antibody Diluent #8112
7. Antigen Unmasking: EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C₁₀H₁₄N₂O₈Na₂•2H₂O) to 1 L dH₂O. Adjust pH to 8.0.
8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
9. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 μl normal goat serum.
10. EnVision+ HRP, Rabbit (Dako North America, Inc. Carpinteria, CA #K4003)
11. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

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 blocking solution for 1 hour at room temperature.
6. Remove blocking solution and add 100–300 μl primary antibody diluted in SignalStain^® Antibody Diluent #8112 to each section. Incubate overnight at 4°C. Refer to product datasheets to determine the recommended dilution: 2085, 3197.
7. Equilibrate EnVision+ reagent to room temperature.
8. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
9. Add 1–2 drops of EnVision+ to each section. Incubate 30 minutes at room temperature.
10. Remove EnVision+ solution and wash sections three times with wash buffer for 5 minutes each.
11. Add 100–400 μl DAB or suitable substrate to each section and monitor staining closely.
12. Upon completion of development, immerse slides in dH₂O.
13. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
14. Wash sections in dH₂O two times for 5 minutes each.
15. 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.
16. Mount coverslips.