Preparation of Reagents:
Normal Goat Serum Blocking Solution
20 ml normal goat serum (filter sterilize with 0.2 µM filter)
Volume to 100 ml of PBS or TBS
Store at 2-8 °C, discard after 3 months
IHC Antibody Diluent (50mM TBS, 1% BSA)
190 ml 1X TBS
2 g BSA (Sigma A9647)
Volume to 200 ml with TBS.
Store 2-8 °C, discard after 1 year.
*May want to add a perservative.
Wash Solution
Can be any of the following: PBS, TBS or 0.01% Tween 20 in dH20. TBS wash solution is recommended for phospho antibodies.
Secondary Antibody
Goat anti-Rabbit IgG Antibody-Dylight conjugated. (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4) Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Protect from light. Optimal working dilutions should be determined experimentally by the investigator.
Procedure:
- Xylene - 3 changes for 5 minutes each
- 100% reagent alcohol - 3 changes for 5 minutes each
- dH20 rinse
- Circle section with a hydrophobic barrier pen. Do not allow sections to dry for the remaining procedure.
- Wash Solution - 3 changes for 5 minutes each
- Normal Goat Serum Blocking Solution - 15 minutes
- Primary Antibody Incubation: 1 hour room temperature. Prepare primary antibody with IHC Antibody Diluent. Optimal working dilutions should be determined experimentally by the investigator. Refer to Bethyl Antibody datasheet for suggested dilution range.
- Wash Solution - 3 changes for 5 minutes each
- Secondary Antibody Incubation: 1 hour room temperature. Protect from light.
- Wash Solution - 3 changes for 5 minutes each
- Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.
NOTE: This procedure is suitable for Phospho-specific Antibodies
