After a subsequent wash with PBS, the cells on the chips were permeabilized using 0.25% Triton X-100 in PBS for 15 min. To block unspecific binding of the antibodies, the cells were blocked with 1% BSA (bovine serum albumin) in PBS/Tween-20 for 30 minutes.
To avoid nonspecific binding of the second antibody, the cells were blocked with 1% BSA at room temperature for 1 h.
All cells were blocked using 10% heat-inactivated murine serum to avoid unspecific binding.
However, the binding of milatuzumab on B cells was blocked completely and confirmed its specificity.
Moreover, when the site is blocked by binding of another intrabody, VL12.3, turnover of soluble mHDx-1 in living cells is blocked.
The uptake in PSMA+ LNCaP cells was blocked in the presence of 2-PMPA, and both compounds showed no binding to PSMA− PC-3 cells at all.
To prevent non-specific binding, cells were incubated with FcR Blocking Reagent (Miltenyi Biotec).
To block unspecific binding, cells were stained in PBS/BSA containing 10% human IgG (Flebogamma; Grifols, Langen, Germany); to exclude dead cells, either propidium iodide (Sigma-Aldrich, Steinheim, Germany) or diamidino phenylindole (Sigma-Aldrich, Steinheim, Germany) was added immediately before flow cytometry.
To block nonspecific binding, cells were incubated with 0.5% Bovine Serum Albumin, 5% Goat Serum for 15 min, and then incubated with primary antibodies: Monoclonal Mouse Anti-Vimentin (clone Vim3B4 Dako, 1 : 100) and Monoclonal Mouse Anti-Matrix Metalloproteinases (MMP) 1 (clone 3B6 Santa Cruz, 1 : 100) for 1 hour at room temperature.
To block nonspecific antibody binding, cells were first incubated for 15 minutes with anti-mouse FC-RIII antibody and normal mouse serum (Jackson Labs).
