Anti-BDNF antibody [3C11] (ab203573) at 5 µg/ml + CHO supernatant containing BDNF at 10 µl

Secondary
HRP-conjugated goat anti-mouse

Predicted band size: 15 kDa

IHC image of BDNF staining in Normal Human Hippocampus formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab203573, 5µg/ml, 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.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-BDNF antibody [3C11] (ab203573) at 5 µg/ml

Lane 1 : Human hippocampus
Lane 2 : Rat hippocampus
Lane 3 : Mouse hippocampus

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : goat anti-mouse IgG IR-680 (green) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa
Additional bands at: 15 kDa (possible mature (processed) protein), 40 kDa (possible immature (unprocessed))

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 ab203573 (1/1000) overnight at 4°C.  Antibody binding was detected using goat anti-mouse IgG IR-680 (green) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx

Immunofluorescent analysis of proBDNF expressing U2OS cells labeling BDNF with ab203573 at 10 µg/mL (Left hand panel). Goat anti-mouse AlexaFluor488 was used as secondary antibody. For nuclear staining DAPI was used. Right hand panel: Negative control (non-transfected U2OS cells).