Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0. 1% Triton X-100 permeabilized U-2 OS (human bone osteosarcoma cell line) cells labeling Pdx1 with APR04530G at 1/25 dilution, followed by Dylight? 488-conjugated goat anti-rabbit IgG (NK179883) secondary antibody at 1/200 dilution (green). Immunofluorescence image showing cytoskeleton staining on U-2 OS cell line. Cytoplasmic actin is detected with Dylight? 554 Phalloidin (PD18466410) at 1/100 dilution (red). The nuclear counter stain is DAPI (blue).

Overlay histogram showing SH-SY5Y cells stained with APR04530G (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 (APR04530G, 1:25 dilution) for 60 min at 37?C. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight? 488 Conjugated Highly Cross-Adsorbed(OH191631) at 1/400 dilution for 40 min at 37?C. Isotype control antibody (blue line) was rabbit IgG (1μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

Fluorescent confocal image of SH-SY5Y cells stained with Nestin (S1409) antibody. SH-SY5Y cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with APR04530G Nestin (S1409) primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor? 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 μg/ml, 5 min). Note the highly specific localization of the Nestin (S1409) immunosignal to the intermediate filaments, supported by Human Protein Atlas Data (http://www.proteinatlas.org/ENSG00000132688).