Formalin-fixed, paraffin-embedded cow kidney tissue stained for Mineralocorticoid Receptor using ab2774 at 1/200 dilution in immunohistochemical analysis.

Heat mediated Antigen retrieval using Citrate buffer, 10 mM pH 6.0 was used.

ab2774 staining Mineralocorticoid Receptor in hamster CHO K1 cells by Immunocytochemistry/ Immunofluorescence. The cells were formaldehyde fixed and then blocked using TBS, 3% Dried Milk, 0.1 % Triton X-100 for 20 minutes at 22°C. Samples were then incubated with primary antibody at 1/100 for 1 hour at 22°C. The secondary antibody used was a goat anti-mouse IgG conjugated to FITC used at a 1/400 dilution (left hand image). In the right hand image DAPI was used to stain the cell nuclei blue.

Overlay histogram showing HEK293 cells stained with ab2774 (red line). The cells were fixed with 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 (ab2774, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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 slightly decreased signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ab2774 (4µg/ml) staining mineralocorticoid receptor in human pancreas, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of the islets of Langerhans and some weaker staining of the exocrine cells of the pancreas.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human colon carcinoma tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Mineralocorticoid Receptor ab2774 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human tonsil tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Mineralocorticoid Receptor ab2774 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human kidney tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Mineralocorticoid Receptor ab2774 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunofluorescent analysis of Mineralocorticoid Receptor using Mineralocorticoid Receptor Monoclonal antibody (H10E4C9F) ab2774 shows staining in HEK293 cells. Mineralocorticoid Receptor staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Mineralocorticoid Receptor ab2774 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of Mineralocorticoid Receptor using Mineralocorticoid Receptor Monoclonal antibody (H10E4C9F) ab2774 shows staining in HeLa cells. Mineralocorticoid Receptor staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Mineralocorticoid Receptor ab2774 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of Mineralocorticoid Receptor using Mineralocorticoid Receptor Monoclonal antibody (H10E4C9F) ab2774 shows staining in HepG2 cells. Mineralocorticoid Receptor staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Mineralocorticoid Receptor ab2774 at a dilution of 1:20-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody . Images were taken at 60X magnification.