Soak the gel for 15 minutes in transfer buffer, then rinse with DI water.
Soak the membrane for 5 minutes in DI water.
Semi-dry transfer at 10V.
After transfer, rinse the membrane once with PBS, then wash 3× for 5 minutes each with PBS (shaking at 50 RPM).
Block with 1× PBS containing 1% LFDM for 30 minutes at room temperature (shake at 50 RPM).
Rinse with 70 ml of PBS.
Add your primary antibodies and incubate overnight in the fridge (3–5°C).
Wash the membrane 5 times (5 minutes each) with PBST containing 0.1% Tween-20 (use 100 ml, shake at 50 RPM).
Add the Adaptor-Secondary of your choice at 1:5000 dilution and incubate with shake for 30 minutes.
Dip in DI water (or 1x PBS).
Incubate with your secondary antibody for example, HRP-Rb anti-Ch (diluted 1:20,000). Use 5 ml per membrane (6×8 cm). Incubate for 30 minutes to 1 hour at room temperature, shaking at 50 RPM.
Wash 4 times (5 minutes each) with 100 ml of PBST, shaking at 50 RPM.
Wash 6 more times with 100 ml of PBS, shaking at 50 RPM.
Add ECL substrate.
Scan the blot on a C-Digit at high resolution (12 minutes).
IHC-P Protocol
Cut the issue at 5 µm
Drain and Dry
Dewax and Dehydrate
HIER using Thermo's PT Module - HIER buffer: Tris-EDTA pH 9.00