Immunocytochemistry/ Immunofluorescence - Aryl hydrocarbon Receptor antibody [RPT9] (ab2769)

ICC/IF image of ab2769 stained HepG2 cells. The cells were 5% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2769, in a 1/200 dilution) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ChIP - Aryl hydrocarbon Receptor antibody [RPT9] (ab2769)

This was a trial experiment to evaluate the association of AhR to the ABCG2 promoter using ab2769 at a 1/100 dilution for the IP (ChIP assay). Semiquantitative PCR was performed to evaluate the relative association of AhR with the proximal ABCG2 promoter in a S1 colon cancer cell line without treatment, or treated with depsipeptide (10 ng/mL 24h) or benzo(a)pyrene (10uM 24h). Cross-linking (X-ChIP) - 10 Mins 0 Secs
Lane 1: DNA ladder (100 bp from promega)
Inputs (lanes 2-4): S1 no treatment - serial dilution
Inputs (lanes 5-7): S1 treated with depsipeptide - serial dilution
Inputs (lanes 8-10): S1 treated with benzo(a)pyrene - serial dilution
AhR ChIP (lane 11-13): use 1:1 diluted immunoprecipitate for PCR
Lane 11=S1 no treatment lane 12=S1 depsipeptide 10ng/mL lane 13= S1 benzo(a)pyrene 10uM
AhR ChIP (lane 14-16): use 1:2 diluted immunoprecipitate for PCR
same order as lanes 11-13
Lane 17: H2O control for PCR.

Image courtesy of an anonymous Abreview.

Flow Cytometry - Aryl hydrocarbon Receptor antibody [RPT9] - ChIP Grade (ab2769)

Overlay histogram showing HEK293 cells stained with ab2769 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2769, 1/20 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.