Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human osteosarcoma cell line) cells labeling RAB23 with BD-PB3855 at 1/25 dilution, followed by Dylight® 488-conjugated goat anti-mouse IgG (174309) secondary antibody at 1/200 dilution (green). Immunofluorescence image showing cytoplasm 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-PB3855(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-Mouse IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed at 1/200 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was mouse IgG1(1μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

All lanes : Anti-RAB23 Antibody at 1:2000 dilution Lane 1: human brain lysate Lane 2: mouse brain lysate Lane 3: MDA-MB-231 whole cell lysate Lane 4: mouse Cerebellum lysate Lane 5: human Cerebellum lysate Lysates/proteins at 20 µg per lane. Secondary Goat Anti-mouse IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 27 kDa Blocking/Dilution buffer: 5% NFDM/TBST.