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.

All lanes : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1 : Mouse GAD67 fragment recombinant protein
Lane 2 : Mouse GAD65 fragment recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 65, 67 kDa

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.

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.

All lanes : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1 : Human cerebellum lysate
Lane 2 : Mouse skin lysate
Lane 3 : Mouse lung lysate
Lane 4 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 6 : Mouse cerebellum lysate
Lane 7 : Rat cerebellum lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 65, 67 kDa
Observed band size: 65,67 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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

Lane 1 : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/5000 dilution
Lane 2 : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 65, 67 kDa
Observed band size: 65,67 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.

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.

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.

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.

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.