Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human osteosarcoma cell line) cells labeling GCA with BD-PB3740 at 1/25 dilution, followed by Dylight® 488-conjugated goat anti-rabbit IgG secondary antibody at 1/200 dilution (green). Immunofluorescence image showing cytoplasm and weak nucleus staining on U-2 OS cell line. Cytoplasmic actin is detected with Dylight® 554 Phalloidin at 1/100 dilution (red).The nuclear counter stain is DAPI (blue).

Overlay histogram showing U-2 OS cells stained with BD-PB3740(green line). The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody for 60 min at 37ºC. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed at 1/200 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was rabbit IgG1 (1μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

All lanes : Anti-GCA Antibody (N-Term) at 1:2000 dilution Lane 1: Human cerebellum lysate Lane 2: Human spleen lysate Lysates/proteins at 20 µg per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 24 kDa Blocking/Dilution buffer: 5% NFDM/TBST.