Specific For: SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit #12692
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Hematoxylin (optional).
NOTE: Additional 10X TBST will be required for washes.
- Tris Buffered Saline with Tween® 20 (TBST-10X) #9997: To prepare wash buffer add 100 μl of Tris Buffered Saline with Tween® 20 (TBST-10X) #9997 to 900 ml of dH2O. OR
- 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. 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.
- Antibody Diluent: SignalStain® Antibody Diluent #8112
- Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: 1X TBST/5% normal goat serum: Add 100 μl 10X TBST (#9997) and 50 μl normal goat serum (#5425) to 850 μl dH2O.
- Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
- Substrate: SignalStain® DAB Substrate Kit (#8059), which includes SignalStain® DAB Diluent (#11724) and SignalStain® DAB Chromogen Concentrate (#11725).
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 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections twice in dH2O for 5 min each.
C. Antigen Unmasking
- Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O twice for 5 min each.
- Wash section in wash buffer for 5 min.
- Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–200 μl primary antibody diluted at 1:500 in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Rabbit (DA1E) mAb XP® SignalStain® Isotype Control (#3900) is used as a negative control. Dilute at 1:500 in SignalStain® Antibody Diluent (#8112) and apply 100–200 μl to each section.
- Equilibrate SignalStain® Boost Detection Reagent (#8114) to room temperature.
- Remove antibody solution and wash sections in wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent (#8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Add 30 μl SignalStain® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain® DAB Diluent (#11724) and mix well before use.
- Apply 100–400 μl SignalStain® DAB to each section and monitor closely. 1–10 minutes generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount coverslips.