Recombinant Anti-Glucocorticoid receptor antibody [41/Glucocorticoid Receptor] (ab302677)

Blocking and diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lysates at 20 µg per lane.

False colour image of Western blot: Anti- Glucocorticoid receptor antibody [41/Glucocorticoid Receptor] (ab302677 staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody 16891 loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab302677 was shown to bind specifically to NR3C1. A band was observed at 94 kDa in wild-type HeLa cell lysates with no signal observed at this size in the NR3C1 knockout cell line. To generate this image, wild-type and NR3C1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat Anti-Mouse IgG H&L (IRDye^® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) (ab216777) at 1/10000 dilution.

Performed under reducing conditions.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

The blot of lane 2 was developed using a high sensitivity ECL substrate.

Exposure time: lane 1: 46 seconds; Lane 2: 37 seconds.

Immunohistochemical analysis of paraffin-embedded human prostatic hyperplasia tissue labeling Glucocorticoid receptor with AB302677 at 1/500 dilution (1.798 µg/ml) followed by ready to use LeicaDS9800 (Bond^® Polymer Refine Detection).

Positive staining on human prostatic hyperplasia is observed.

The section was incubated with ab302677 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human cervical cancer tissue labeling Glucocorticoid receptor with AB302677 at 1/500 dilution (1.798 µg/ml) followed by ready to use LeicaDS9800 (Bond^® Polymer Refine Detection).

Positive staining on human cervical cancer is observed.

The section was incubated with ab302677 for 30 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Glucocorticoid receptor with AB302677 at 1/50 dilution (17.98 µg/mL), followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (Green).

Confocal image showing all the signal translocated from cytoplasm into nucleus in HeLa cells treated with dexamethasone (1 µM) for 20 min.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab202272 Recombinant Alexa Fluor^® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/mL).