Fitc conjugated goat anti mouse igg1

Peripheral blood mononuclear cells were pipetted onto a glass slide previously treated with acetic acid 2 N and 70% ethanol. Adherent cells were fixed in 4% paraformaldehyde in PBS and 0.12 M sucrose for 20 min at room temperature. For the process of permeabilization, cells were treated with 0.2% Triton X-100 in PBS for 20 min.
Non-specific binding sites were blocked using 6% goat serum. Cells were incubated overnight at 4°C with monoclonal anti-BTK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), that recognizes the epitope at amino acid positions 459-659 of the human BTK. Further, we used a FITC-conjugated goat anti-mouse IgG1 as the secondary antibody (SouthernBiotech – 1:1000). After extensive washing with PBS, the coverslips were incubated with Hoechst (Life Technologies) for visualization of the nucleus and mounted on slides using Prolong Gold antifade (Life Technologies). Images were generated in a confocal fluorescence microscope (LSM - 510 Meta, ZEISS, Jena, Germany) with a magnification of 63×.

FACS staining was performed as described recently [7 (link)]. MCs were labeled with primary antibodies anti-CD16-PE (Becton Dickinson, San Jose, CA), anti-CD32-PE, anti-CD64-PE, anti-CD89 (Caltag Laboratories, Hamburg, Germany), FcϵRI α-chain (mAb 22E7, Hoffmann-La Roche, kindly provided by R. Chizzonite), anti-HLA-DR, (DAKO A/S, Glostrup, Denmark) or appropriate isotype controls. Cells were washed and if purified primary Ab were used incubated with FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birminghman, AL). FACS analysis was performed using the FACSCalibur™ system (Becton Dickinson). Analysis were performed using the FlowJo software (Treestar, Inc. Ashland, OR).