Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervical epithelial adenocarcinoma cell line) cells labeling DENR with BD-PB3471 at 1/25 dilution, followed by Dylight® 488-conjugated goat anti-mouse IgG secondary antibody at 1/200 dilution (green). Immunofluorescence image showing cytoplasm staining on HeLa 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 Hela cells stained with BD-PB3471 (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 1/400 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was mouse IgG2b (1μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

BD-PB3471 staining DENR in human skeletal muscle sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 0. 5 hour at room temperature; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

All lanes : Anti-DENR Antibody at 1:1000-1:2000 dilution Lane 1: A431 whole cell lysate Lane 2: human skeletal muscle lysate Lane 3: human brain lysate Lysates/proteins at 20 µg per lane. Secondary Goat Anti-mouse IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 22 kDa Blocking/Dilution buffer: 5% NFDM/TBST.