#2197 Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb用プロトコール
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%)
- Deionized water (dH2O)
- Hematoxylin (optional)
- Wash Buffer:
- 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.
- SignalStain® Antibody Diluent (#8112).
- Antigen Unmasking Citrate: 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: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
- Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
- Substrate: SignalStain® DAB Substrate Kit (#8059).
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
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; 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 minutes each.
- Incubate sections in 3% hydrogen peroxide for 10 minutes.
- Wash sections in dH2O twice for 5 minutes each.
- Wash sections 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 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Prepare ABC solution per manufacturer's recommendations.
- Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
- Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
- Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
- Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
- Wash section three times with wash buffer for 5 min each.
- Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent 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 dH20.
- 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.
posted April 2015