Anti-BDNF antibody [EPR1292](AB108319)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with unpurified ab108319 (1/1000) overnight at 4°C. ab8245 (mouse anti-GAPDH; 0.05 ug/mL) was included as a loading control. Antibody binding was detected using goat anti-rabbit IgG IR-680 (green) and goat anti-mouse IgG IR800 (red) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx

All lanes: Western blot - Anti-BDNF antibody [EPR1292] (ab108319) at 1/1000 dilution

Lane 1: Human hippocampus lysate at 20 µg

Lane 2: Rat hippocampus lysate at 20 µg

Lane 3: Mouse hippocampus lysate at 20 µg

Secondary

All lanes: Gt anti Rb IR680 at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 27 kDa

Observed band size: 15 kDa, 28 kDa, 35 kDa, 45 kDa

All lanes: Western blot - Anti-BDNF antibody [EPR1292] (ab108319) at 1/1000 dilution

Lane 1: Human brain lysates at 20 µg

Lane 2: Mouse brain lysates at 20 µg

Lane 3: Rat brain lysates at 20 µg

Lane 4: Human hippocampus lysates at 20 µg

Lane 5: Mouse hippocampus lysates at 20 µg

Lane 6: Rat hippocampus lysates at 20 µg

Lane 7: Human cerebellum lysates at 20 µg

Lane 8: Mouse cerebellum lysates at 20 µg

Lane 9: Rat cerebellum lysates at 20 µg

Secondary

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

Predicted band size: 27 kDa

Observed band size: 15-45 kDa

IHC image of BDNF staining in a section of frozen normal human cerebral cortex performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab108319, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunohistochemistry (Frozen sections) analysis of rat cerebral cortex tissue sections labeling BDNF with Purified ab108319 at 1/100 (2.8 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder cancer tissue sections labeling BDNF with Purified ab108319 at 1:500 dilution (0.56 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BDNF with purified ab108319 at 1/30 dilution (10μg/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluorr® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).