After washing and blocking, the antibody solution (1 10,000 diluted Anti-Digoxigenin-AP (Roche) in blocking solution) was added to the membrane.
"But, obviously, blocking the service is not the solution".
After removal of the blocking solution, the membrane was incubated with a fresh blocking solution containing the primary antibody at a specific dilution (1∶10000 to 1∶25000) for 1 hour.
After removal of the blocking solution the wells were washed 3 times for 5 min each at 25°C with PBST.
After aspiration of the blocking solution, the plates were sealed and stored at 4 °C until use.
After removing the blocking solution, the sections were incubated with a goat anti-human IgM diluted 1 100 (μ chain-specific, Vector Laboratories, Burlingame, California, USA, code FI-3020 ).
Drain the blocking solution from the slides and apply two drops of avidin solution (Avidin biotin blocking kit; Vector Laboratories; Dossenheim-Germany; SP2001) for 15 min.
Plates were incubated for 1 h at room temperature with the G12 moAb (1∶1,000 in the blocking solution), A1 moAb (1∶500 in the blocking solution) or 6B5L1 moAb (1∶1,000 in the blocking solution).
The membrane was then incubated with the blocking solution containing the first antibody overnight at 4°C.
The sections were then incubated with the blocking solution containing the primary antibody, at 4 °C overnight in a moist box.
Following their incubation in blocking solution, the slides were incubated with the CM2B4 antibody diluted 1 : 100.
