Anti-rabbit IgG, the secondary Ab (1 2000 dilution in the blocking solution), was added, followed by incubation for 1 h at RT, then blots were again washed three times with TBST and reacted to BCIP-NBT solution (Nakanai tesque, Japan).
Then, 100 μl of serum blocking solution was added to each section, followed by incubation for 10 min and then draining the solution.
To detect recombinant I329L, a primary rat monoclonal high-affinity antibody against HA diluted in blocking solution was used (1 h, RT), followed by a donkey affinity-purified anti-rat IgG antibody conjugated with AMCA (1 h, RT).
Antibody solution (anti-Digoxigenin-AP, Fab fragments from Roche diluted 1 4000 in blocking solution) was incubated ON at 4°C, followed by 3×15 min washes in blocking solution and 3×15 min in NTMT (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 0.05 M MgCl, 1 mM Levamisol, 0.1% Tween-20).
Fcγ receptor blocking solution was placed on the tissue, followed by 15 min incubation with biotinylated rabbit anti-collagen IV (1 μg/ml; Abcam, Cambridge, MA USA), a brief rinse with Ringer's solution and 15 min incubation with streptavidin-Pacific Blue (1 μg/ml; Invitrogen, Zug, Switzerland).
Following three washes of D-PBS, 50 μl/well of primary antibodies at 2 μg/ml in blocking solution were added for 1 h at room temperature (RT).
Blocking solution was applied for 20 min, followed by incubation with the primary antibody (1 : 10 dilution TBS) for 30 min.
Blocking solution was removed and primary antibody in blocking solution applied at the required dilution.
Blocking solution was used to block the endogenous peroxidase activity.
The sections were incubated with a rat monoclonal anti-CD31 (PECantibodyibodilutedted 1 200 in blocking solution at 4°C, which was followed by incubation with Alexa Fluor® 555-conjugated rabbit anti- rat IgG) diluted 1:1000 in blocking solution.
