重组Anti-GABA A Receptor alpha 2/GABRA2抗体[EPR26485-185] (ab307359)

All lanes : Anti-GABA A Receptor alpha 2/GABRA2 antibody [EPR26485-185] (ab307359) at 1/1000 dilution

Lane 1 : Mouse cerebellum tissue lysate 80 µg
Lane 2 : Mouse brain tissue lysate 80 µg
Lane 3 : Mouse lung tissue lysate 80 µg
Lane 4 : Mouse liver tissue lysate 80 µg
Lane 5 : Rat cerebellum tissue lysate 80 µg
Lane 6 : Rat brain tissue lysate 80 µg
Lane 7 : Rat lung tissue lysate 80 µg

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

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

Low expression: liver, lung (PMID:29467616)

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

Exposure time: 48 seconds

Anti-GABA A Receptor alpha 2/GABRA2 antibody [EPR26485-185] (ab307359) at 1/1000 dilution + human hippocampus tissue lysate 40 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

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

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

Exposure time: 48 seconds

All lanes : Anti-GABA A Receptor alpha 2/GABRA2 antibody [EPR26485-185] (ab307359) at 1/1000 dilution

Lane 1 : Mouse hippocampus tissue lysate 20 µg
Lane 2 : Rat hippocampus tissue lysate 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

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

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

Exposure time: 5.5 seconds

Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/2000 dilution (0.241 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Positive staining on mouse hippocampus (PMID: 23337532).
The section was incubated with ab307359 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 ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/2000 dilution (0.241 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Positive staining on rat hippocampus (PMID: 23337532).
The section was incubated with ab307359 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 ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/2000 dilution (0.241 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Negative control: No staining on mouse lung. 
The section was incubated with ab307359 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 ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed 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 hippocampus (fresh) tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/mL) (Green).
Confocal image showing positive staining on mouse hippocampus.
The section was incubated with ab307359 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). 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 hippocampus (fresh) tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/mL) (Green).
Confocal image showing positive staining on rat hippocampus.
The section was incubated with ab307359 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). 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 lung (fresh) tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/mL) (Green).
Low expression: confocal image showing no staining on mouse lung (PMID: 29467616). 
The section was incubated with ab307359 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). 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  lung (fresh) tissue labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/mL) (Green).
Low expression: confocal image showing no staining on rat lung (PMID: 29467616). 
The section was incubated with ab307359 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). 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).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary neurons labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 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 cytoplasmic staining in mouse primary neurons.
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (4 µg/ml), followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (2 µg/ml) (Red). Nuclear counterstain was DAPI (Blue).

-ve control 1: AB307359 at a 1/500 dilution followed by ab150120 at a 1/1000 diliution.
-ve control 2: ab11267 at a 1/500 dilution followed by ab150081 at a 1/1000 diliution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized rat hippocampal neurons labeling GABA A Receptor alpha 2/GABRA2 with ab307359 at 1/100 dilution (4.81 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 cytoplasmic staining in rat hippocampal neurons.
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (4 µg/ml), followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (2 µg/ml) (Red). Nuclear counterstain was DAPI (Blue).

-ve control 1: AB307359 at a 1/500 dilution followed by ab150120 at a 1/1000 diliution.
-ve control 2: ab11267 at a 1/500 dilution followed by ab150081 at a 1/1000 diliution.

GABA A Receptor alpha 2/GABRA2 was immunoprecipitated from 0.35 mg mouse cerebellum tissue lysate 10 µg with ab307359 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307359 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse cerebellum tissue lysate 10 µg

Lane 2: ab307359 IP in mouse cerebellum tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307359 in mouse cerebellum tissue lysate

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

Exposure time: 24 seconds.