All lanes : Anti-GAD65 antibody [EPR22952-70] (ab239372) at 1/5000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse cerebellum tissue lysate
Lane 3 : Rat brain tissue lysate
Lane 4 : Rat cerebellum tissue 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 kDa

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

Exposure time: 8 seconds.

Bands above 100kDa are multimers of GAD65.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 10601283, 21734072).

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat pancreas tissue labeling GAD65 with ab239372 at 1/100 dilution (Green) followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution. The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution.

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling GAD65 with ab239372 at 1/2000 (0.323 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on islet of rat pancreas. The section was incubated with ab239372 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Mouse cerebellum tissue labeling GAD65 with ab239372 at 1/100 dilution (Green) followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution. The nuclear counterstain was DAPI (Blue). Positive staining on mouse cerebellum is observed.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling GAD65 with ab239372 at 1/2000 (0.323 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum is observed. The section was incubated with ab239372 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling GAD65 with ab239372 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in mouse primary neurons.is observed. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue). Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Beta-TC-6 (Mouse pancreas insulinoma beta cell) cells labelling GAD65 with ab239372 at 1/60 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

GAD65 was immunoprecipitated from 0.35 mg Mouse brain lysate with ab239372 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab239372 at 1/1000 dilution (0.45 μg/ml). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain lysate 10 μg

Lane 2: ab239372 IP in Mouse brain lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239372 in Mouse brain lysate.

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

Exposure time: 3 seconds

Bands above 100kDa are multimers of GAD65 (PMID: 10601283).