After blocking with PBS containing 0.05% Tween 20 and 0.25% bovine serum albumin (BSA), pH 7.4 (blocking buffer), serial dilutions of patient serum in blocking buffer were incubated for 4 h at room temperature.
After three washes and 5 min blocking in blocking buffer, samples were incubated with a polyclonal anti-Neisseria antibody (diluted 1∶100 in blocking buffer) for 1 hour, to detect intracellular and extracellular bacteria.
Plasma samples diluted 1 2,500 with dilution buffer (30 μL/well) or standards (30 μL/well) along with 10 μL of biotinylated antigens in blocking buffer were incubated with 10 μl of pre-mixed bead sets into the wells of a pre-wet 96-well microtitre plate.
Primary antibodies diluted 1 1000 in Odyssey Blocking Buffer were incubated with membranes at room temperature for 2 h.
After removing blocking buffer, samples were incubated with primary antibody in blocking buffer overnight at 4°C and then washed three times with IF wash buffer, followed by incubating with Alexa Fluor 488-conjugated secondary antibody (Life technologies) for 40 minutes in a dark humidified chamber and then washed three times with IF wash buffer for 20 minutes.
After addition of the stop solution and blocking buffer, sections were incubated with 1 × conjugate solution for 30 min at RT, and the TUNEL-positive cells were visualized using a DAB kit (Calbiochem).
After incubation for 60 min in blocking buffer, cells were incubated with primary antibodies for 60 min at room temperature or overnight at 4°C (primary antibody IgG was diluted into PBS with 0.05% Triton X-100 and 0.2% BSA).
After washing again with PBTx and incubation for 1 hour in block buffer, embryos were incubated in the second antibody overnight at 4°C.
After removal of blocking buffer, sections were incubated at 4°C overnight with fluorescein-reporter conjugated and Biotin conjugated c-Met antibodies prepared in blocking buffer.
