*IMPORTANT: See product data sheets for the appropriate antibody dilution.
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%)
- Deionized water (dH2O)
- Hematoxylin (optional)
- 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.
- Antibody Diluent: SignalStain® Antibody Diluent #8112
- 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.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 μl normal goat serum.
- EnVision™+ HRP, Rabbit (Dako North America, Inc. Carpinteria, CA #K4003)
- DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 minutes each.
- Incubate sections in two washes of 100% ethanol for 10 minutes each.
- Incubate sections in two washes of 95% ethanol for 10 minutes each.
- 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.
- Place slides in 250 ml room temperature EDTA unmasking solution in a 24-slide holder.
- Place 500 ml dH2O into the pressure cooker.
- 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.
- Seal the chamber and proceed with retrieval. Settings for the Biocare Medical Decloaking Chamber follow.
- SP1 125°C 30 seconds
- SP2 90°C 10 seconds
- Carefully vent the device, then remove the lid and cool the slides on the bench for 10 minutes.
- Rinse the slides with dH2O.
- Wash sections in dH2O three times for 5 minutes each.
- Incubate sections in 3% hydrogen peroxide for 10 minutes.
- Wash sections in dH2O twice for 5 minutes each.
- Wash section in wash buffer for 5 minutes.
- Block each section with 100–400 μl blocking solution for 1 hour at room temperature.
- 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.
- Equilibrate EnVision™+ reagent to room temperature.
- Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
- Add 1–2 drops of EnVision™+ to each section. Incubate 30 minutes at room temperature.
- Remove EnVision™+ solution and wash sections three times with wash buffer for 5 minutes each.
- Add 100–400 μl DAB or suitable substrate to each section and monitor staining closely.
- Upon completion of development, immerse slides in dH2O.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 minutes each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 seconds each.
- Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
- Repeat in xylene, incubating sections two times for 10 seconds each.
- Mount coverslips.