Anti-KCC2 antibody [EPR24203-85]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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 Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

All lanes:

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

Predicted band size: 126 kDa

Observed band size: 140 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-KCC2 antibody [EPR24203-85] (AB259969)

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).

All lanes:

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

Predicted band size: 126 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - 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.

Exposure time : 15 seconds

All lanes:

Western blot - Anti-KCC2 antibody [EPR24203-85] (ab259969) at 1/1000 dilution

All lanes:

Human spinal cord at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/5000 dilution

Predicted band size: 126 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - 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 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

All lanes:

Western blot - Anti-KCC2 antibody [EPR24203-85] (ab259969) at 1/1000 dilution

All lanes:

Mouse spinal cord tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 126 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - 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

All lanes:

Western blot - Anti-KCC2 antibody [EPR24203-85] (ab259969) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 126 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - 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

All lanes:

Western blot - Anti-KCC2 antibody [EPR24203-85] (ab259969) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat spinal cord tissue lysate at 20 µg

Lane 3:

Rat kidney tissue lysate

Lane 4:

Rat liver tissue lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 126 kDa

Observed band size: 140 kDa

false