Recombinant Anti-KCC2 antibody [EPR24203-85] (ab259969)

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

Samples are non-boiled as boiling may cause protein aggregates.

The observed 30 KD band is like the KCC2 C terminus fragment which has been described in the literature (PMID: 22854961).

Negative control: Kidney, liver (PMID: 10212246).

Exposure time: 6 seconds

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

Samples are non-boiled as boiling may cause protein aggregates.

The observed 30 KD band is like the KCC2 C terminus fragment which has been described in the literature (PMID: 22854961).

Negative control: Kidney, liver (PMID: 10212246).

Exposure time: 6 seconds

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

Samples are non-boiled as boiling may cause protein aggregates.

The observed 23, 30 KD bands are like the KCC2 C terminus fragment and the observed 300 KD band is the dimer KCC2, as described in the literature (PMID: 22854961).

Exposure time: 26 seconds

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

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: 15 seconds

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labelling KCC2 with ab259969 at 1/5000 dilution (0.111 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human cerebrum.The section was incubated with ab259969 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labelling KCC2 with ab259969 at 1/5000 dilution (0.111 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum (PMID: 17715129). The section was incubated with ab259969 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling KCC2 with ab259969 at 1/5000 dilution (0.111 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab259969 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labelling KCC2 with ab259969 at 1/5000 dilution (0.111 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab259969 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Negative control: almost no staining on mouse liver.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum (fresh) tissue labeling KCC2 with ab259969 at 1/100 dilution (5.55 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml)(Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue).

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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum (fresh) tissue labeling KCC2 with ab259969 at 1/100 (5.55 µg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml)(Green). Positive staining on rat cerebrum is observed. The nuclear counterstain was DAPI (Blue).

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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver(fresh) tissue labeling KCC2 with ab259969 at 1/100 dilution (5.55 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml)(Green). The nuclear counterstain was DAPI (Blue). No staining on mouse liver.

Negative control: liver (PMID:17715129) is observed.

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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver (fresh) tissue labeling KCC2 with ab259969 at 1/100 dilution (5.55 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml)(Green). The nuclear counterstain was DAPI (Blue). No staining on rat liver. 

Negative control: liver (PMID:17715129) is observed.

Secondary antibody 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 mouse primary neural/glia cells labelling KCC2 with ab259969 at 1/200 dilution (2.775 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml)(Green). ab11267 Anti-MAP2 mouse monoclonal antibody at 1/500 dilution (4 µg/ml) followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) antibody at 1/1000 dilution (2 µg/ml) was used to counterstain tubulin (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing positive staining in mouse primary neuron. Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

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).

Negative control 1: ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) antibody at 1/1000 dilution (2 µg/ml).

Negative control 2: ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (4 µg/ml).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neural/glia cells labelling KCC2 with ab259969 at 1/200 dilution (2.775 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml)(Green). ab11267 Anti-MAP2 mouse monoclonal antibody at 1/500 dilution (4 µg/ml) followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) antibody at 1/1000 dilution (2 µg/ml) was used to counterstain tubulin (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing positive staining in rat primary neuron. Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

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).

Negative control 1: ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) antibody at 1/1000 dilution (2 µg/ml).

Negative control 2: ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (4 µg/ml). 

Flow cytometric (intracellular) analysis of 4% paraformaldehyde-fixed Mouse primary neuron cells labelling KCC2 with ab259969 at 1/500 dilution (Right) compared with Isotype control Rabbit monoclonal IgG (ab172730) (Left). Goat Anti-Rabbit IgG Alexa Fluor® 488 (ab150081) was used as the secondary antibody.

Flow cytometric (intracellular) analysis of 4% paraformaldehyde-fixed Rat primary neuron cells labelling KCC2 with ab259969 at 1/500 dilution (Right) compared with Isotype control Rabbit monoclonal IgG (ab172730) (Left). Goat Anti-Rabbit IgG Alexa Fluor® 488 (ab150081) at 1/2000 dilution was used as the secondary antibody.

KCC2 was immunoprecipitated from 0.35 mg mouse brain tissue lysate with ab259969 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259969 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10µg

Lane 2: ab259969 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259969 in mouse brain tissue lysate

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

Exposure time: 3 seconds

The observed 30 KD band is like the KCC2 C terminus fragment and the observed 300 KD band is the dimer KCC2, as described in the literature (PMID: 22854961).

KCC2 was immunoprecipitated from 0.35 mg rat brain tissue lysate with ab259969 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259969 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Rat brain tissue lysate 10µg

Lane 2: ab259969 IP in Rat brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259969 in rat brain tissue lysate

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

Exposure time: 3 seconds

The observed 30 KD band is like the KCC2 C terminus fragment and the observed 300 KD band is the dimer KCC2, as described in the literature (PMID: 22854961).