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. 2014 Apr 11;8:107. doi: 10.3389/fncel.2014.00107

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Copyright © 2014 Andreska, Aufmkolk, Sauer and Blum.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Two anti-BDNF antibodies enable specific and dense indirect immunolabeling of endogenous BDNF in hippocampal neurons in vitro. (A) Antibody verification with recombinant BDNF. HeLa cells stably expressing lentivirally-delivered BDNF^IRESGFP were labeled with different anti (α)-BDNF antibodies from different species (r, rabbit; m, mouse; s, sheep). Antibody details are given in the Material and Method section. As transduction control, GFP was expressed bicistronically under an IRES2 sequence and postlabeled with chicken anti-GFP. BDNF was accumulated in the early secretory trafficking pathway after microtubule disruption with nocodazole, causing a perinuclear Golgi-like staining pattern. (B) Immunoblot of rαBDNF (17 h). Serial dilutions of recombinant BDNF (250–31 pg per lane) served as control (left panel). Hippocampal BDNF harvested at postnatal day 5 was absent in bdnf^−/− mice, while heterozygous and wildtype controls show mature BDNF at a M_r of 13 kDa. HRP-conjugated anti-Cytochrome C (Cyt C) served as loading control (left panel). In adult mice, rαBDNF (17 h) revealed mature BDNF at high concentration in the hippocampus (left and right lane), while only low amounts were detected in cerebellar protein lysates (middle lane). (right panel). Immunoblots revealed high specificity of the antibody 17 h. (C) Endogenous BDNF in hippocampal neurons at day in vitro (DIV) 35. In somatic regions, BDNF was not concentrated in perinuclear Golgi cisternae, but rather in somatic vesicles. Golgi cisternae were labeled with luminal GFP, a marker for the anterograde, secretory pathway. Luminal GFP and endogenous BDNF co-labeled anterograde trafficking structures (arrow heads) in neurites of hippocampal neurons. Endogenous BDNF was double-labeled by two different anti-BDNF antibodies (rabbit anti-BDNF 17h and mouse anti-BDNF mab9). (D) Somatic anti-BDNF labels were lost when the bdnf gene was removed from single cells. Hippocampal neurons from bdnf^fl/fl mice were transduced with a low titer of a lentivirus expressing tdTomato^IRESCre. Neurons (DIV 20) were labeled with anti-BDNF (mab9). Tomato+ cells (upper neuron, red, yellow arrow) lacked a typical vesicular somatic BDNF label, while untransduced, Tomato-Cre-deficient neurons (lower neuron, cyan arrow) exhibited a strong somatic BDNF label. In the composite image Tomato (red) and mouse anti-BDNF (mab9) (green) labels are shown together with an anti-neurofilament (Nfh) label (white) and a nuclear DAPI stain (blue). Bar: 25 μm.