Recombinant Anti-ATP1A1+ATP1A2+ATP1A3+ATP1A4 antibody [EPR25414-249] (ab300507)

Blocking / Diluting buffer and concentration:5% NFDM/TBST

Note: The samples are unboiled

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

Exposure time: Lanes 1-3: 3 seconds; lane 4: 15 seconds.

Note: The samples are unboiled.

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

The samples are unboiled.

Note: Bands above 110 kDa are aggregates of Na+/K+-ATPase alpha subunits.

Immunohistochemical analysis of paraffin-embedded human cardiac muscle labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1:5000 (0.108 μg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining on human cardiac muscle, particularly on the intercalated discs (PMID: 26109061). The section was incubated with ab300507 for 30 mins at room temperature. 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 cerebrum labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1:5000 (0.108 μg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining on human cerebrum. The section was incubated with ab300507 for 30 mins at room temperature. 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 mouse cerebrum labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1:5000 (0.108 μg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining on mouse cerebrum (PMID: 32301109) . The section was incubated with ab300507 for 30 mins at room temperature. 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 rat cardiac muscle labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1:5000 (0.108 μg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining on rat cardiac muscle, particularly on the intercalated discs. The section was incubated with ab300507 for 30 mins at room temperature. 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 rat cerebrum labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1:5000 (0.108 μg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Positive staining on rat cerebrum. The section was incubated with ab300507 for 30 mins at room temperature. 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 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen sectioned mouse cerebrum (fresh) labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) at 1/1000 dilution (green). Positive staining on mouse cerebrum is observed. The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) at 1/1000 dilution.

No antigen retrieval was carried out.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen sectioned rat cerebrum (fresh) labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) at 1/1000 dilution (green). Positive staining on rat cerebrum is observed. The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) at 1/1000 dilution.

No antigen retrieval was carried out.

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HEK293T (human embryonic kidney epithelial cell) labelling ATP1A3 with ab300507 at 1/500 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Anti -Myc-Tag Mouse mAb (Alexa Fluor^® 647) was used as a counterstain at 1/100 dilution (0.38μg/m) (red). Confocal image showing cytoplasmic and membranous staining in HEK-293T cells transfected with human ATP1A3 expression vector containing a myc tag. The nuclear counter stain is DAPI (blue).

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HEK293T (human embryonic kidney epithelial cell) labelling ATP1A1+ATP1A2+ATP1A4 with ab300507 at 1/500 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Anti -Myc-Tag Mouse mAb (Alexa Fluor^® 647) was used as a counterstain at 1/100 dilution (0.38μg/m) (red). Confocal image showing cytoplasmic and membranous staining in HEK-293T cells transfected with human ATP1A1 or ATP1A2 or ATP1A4 expression vector containing a myc tag. The nuclear counter stain is DAPI (blue).

Immunofluorescent analysis of 100% methanol fixed, 0.1% Triton X-100 permeabilized A-172 (human brain glioblastoma) labelling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing membranous and weak cytoplasmic staining in A-172 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

Immunofluorescent analysis of 100% methanol fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblasts) labelling ATP1A1+ATP1A2+ATP1A3+ATP1A4 with ab300507 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing membrane and weak cytoplasm staining in Neuro-2a cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

Flow Cytometry (Intracellular) analysis of A-172 (human brain glioblastoma) labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 (red) with ab300405 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies. 

Flow Cytometry (Intracellular) analysis of Neuro-2a (mouse neuroblastoma neuroblasts) labeling ATP1A1+ATP1A2+ATP1A3+ATP1A4 (red) with ab300405 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies.