Anti-GAD65 + GAD67 antibody [EPR19366]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded Mouse cerebellum tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse cerebellum is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

ab183999 Immunoprecipitating GAD65 + GAD67 in Human cerebellum lysate. 10μg of cell lysate was incubated with primary antibody 1/40. For western blotting ab183999 (1/1000) was used to confirm successful immunoprecipation. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000.

Lane 1 (Input) : Human cerebellum lysate 10μg
Lane 2 (+) : Human cerebellum lysate with ab183999, 1/40
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab183999 in Human cerebellum lysate

All lanes:

Immunoprecipitation - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

Predicted band size: 65 kDa

false

Exposure time: 2min

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat pancreas islet is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse pancreas islets is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative on rat kidney. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative on mouse lung. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of paraffin-embedded Rat cerebellum tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat cerebellum is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunohistochemical analysis of 4% paraformaldehyde fixed, 0.2% Triton X-100 permealized frozen section of Mouse hippocampus tissue labeling GAD65 + GAD67 with ab183999 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) secondary (ab150077) at 1/1000 dilution (green). The result showed mainly cytoplasmic staining on mouse hippocampus. The nuclear counterstain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Immunocytochemistry/ Immunofluorescence analysis of mouse primary neuron cells labeling GAD65 + GAD67 with purified ab183999 at 1/100 (10 μg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab11267 Anti-MAP2 antibody [HM-2]; ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

• WB

Supplier Data

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-5 : 3 minutes; Lane 6 and 7 : 2 seconds.

All lanes:

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1:

Human cerebellum lysate at 20 µg

Lane 2:

Mouse skin lysate at 20 µg

Lane 3:

Mouse lung lysate at 20 µg

Lane 4:

Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate at 20 µg

Lane 5:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Lane 6:

Mouse cerebellum lysate at 20 µg

Lane 7:

Rat cerebellum 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: 65 kDa

Observed band size: 65 kDa,67 kDa

false

• WB

Supplier Data

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Blocking/Dilution buffer : 5% NFDM/TBST.

Lane 1:

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/5000 dilution

Lane 2:

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Rat brain lysate at 10 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: 65 kDa

Observed band size: 65 kDa,67 kDa

false

Exposure time: 2s

• WB

Supplier Data

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (AB183999)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 1 second; Lane 2 : 15 seconds.

Mouse GAD67 fragment recombinant protein contain aa468-592 with a His-Tag®. Mouse GAD65 fragment recombinant protein contain aa460-584 with a His-Tag®. These two fragment recombinant proteins were made in-house. The ~30 kDa band represents doublets of the recombinant fragments.

All lanes:

Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1:

Mouse GAD67 fragment recombinant protein at 0.01 µg

Lane 2:

Mouse GAD65 fragment recombinant protein at 0.01 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: 65 kDa

false