Recombinant Anti-NKCC1 antibody [EPR26256-60] (ab303518)

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

This antibody does not cross-react with mouse NKCC2.

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

Low expression: liver (human protein atlas database)

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

NKCC1 was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518 at dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: mouse brain tissue lysate 10 µg (Inset)

Lane 2: ab303518 IP in mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab303518 in mouse brain tissue lysate

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

Exposure time: 15 seconds.

NKCC1 was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518 at dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: rat brain tissue lysate 10 µg (Inset)

Lane 2: ab303518 IP in rat brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab303518 in rat brain tissue lysate

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

Exposure time: 23 seconds.

NKCC1 was immunoprecipitated from 0.35 mg human cerebellum tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518 at dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: human cerebellum tissue lysate 10 µg (Inset)

Lane 2: ab303518 IP in human cerebellum tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab303518 in human cerebellum tissue lysate

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

Exposure time: 3 minutes

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling NKCC1 with ab303518 at 1/100 dilution (4.89 µg/mL), followed by a ready to use LeicaDS9800 dilution (BOND^™ Polymer Refine Detection). Positive staining was observed on human colon (PMID: 17478539). The section was incubated with ab303518 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 dilution (BOND^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling NKCC1 with ab303518 at 1/2000 dilution (4.89 µg/mL), followed by a ready to use LeicaDS9800 dilution (BOND^™ Polymer Refine Detection). Positive staining was observed on mouse colon. The section was incubated with ab303518 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 dilution (BOND^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling NKCC1 with ab303518 at 1/2000 dilution (4.89 µg/mL), followed by a ready to use LeicaDS9800 dilution (BOND^™ Polymer Refine Detection). Positive staining was observed on rat colon. The section was incubated with ab303518 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 dilution (BOND^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling NKCC1 with ab303518 at 1/100 dilution (4.89 µg/mL), followed by a ready to use LeicaDS9800 dilution (BOND^™ Polymer Refine Detection). Negative control: no staining is observed on human liver. The section was incubated with ab303518 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T84 (human colon epithelial cell) cells labeling NKCC1 with ab303518 at 1/100 dilution (4.89 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing mainly membranous staining in T84 cell line. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/mL).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling NKCC1 with ab303518 at 1/100 dilution (4.89 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing membranous and cytoplasmic staining in NIH/3T3 cell line. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/mL).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized T84 (human colon epithelial cell) cells labeling NKCC1 with ab303518 at 1/50 dilution (1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling NKCC1 with ab303518 at 1/50 dilution (1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.