Anti bdnf

The neural retina was detached from the RPE, and both tissues were homogenized as previously described [13 (link), 14 (link), 34 (link)]. Proteins (50 μg/sample) were separated in SDS, 12% polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and incubated overnight at 4 °C with a mouse monoclonal anti-cytochrome c (1:1000, Santa Cruz Biotechnology, Dallas TX, USA), a rabbit polyclonal anti-TOM20 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-VDAC (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), a mouse polyclonal anti-RPE65 (1:500; EMD Millipore, Darmstadt, Germany, MAB5428) and a mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). The following secondary antibodies were used: a donkey anti-mouse (1:2000, Jackson Laboratory, Bar Harbor, ME, USA) and a donkey anti-rabbit (1:2000, Jackson Laboratory, Bar Harbor, ME, USA). Densitometric signals were quantified using ImageQuant software and adjusted by the density of β-actin. For each group, the mean of five homogenates were averaged and taken as the representative value.

Primary antibodies used for immunoblotting were as follows: anti-IKAP (Anaspec, cat# 54494), anti-acetylated α-tubulin (Sigma, cat# t7451), anti-BDNF (Alomone Labs, cat# ANT010), and anti-Hsc70 (Santa Cruz Biotechnology, cat# sc-7298). Secondary antibodies were donkey anti-rabbit IgG HRP (Abcam, cat# ab97064), or donkey anti-mouse IgG HRP (Abcam, cat# ab98799), as appropriate.

Western blot was performed as previously described [45 (link)]; briefly HPC and PFC were lysed in ice-cold RIPA buffer (0.15 mM NaCl, 0.05 mM Tris_HCl, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) with Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy). Protein concentration was determined by the Quantum Bicinchoninic Protein Assay (Euroclone) and 15 μg of proteins was separated on SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Amersham, Milan, Italy), followed by blocking in 5% milk dissolved in Tris Buffer Saline-Tween 20 (TBST). Membranes were incubated with anti-BDNF (1 : 2,000, rabbit polyclonal, Alomone, Jerusalem, Israel) or anti-β-actin (1 : 20,000, mouse monoclonal, Sigma) antibodies. After washing with TBST, filters were incubated with peroxidase-conjugated secondary anti-rabbit (1 : 3,000, Sigma-Aldrich) or with the fluorescent IRDye secondary anti-mouse antibody (LI-COR, purchased from Carlo Erba Reagents, Milan, Italy). Peroxidase immunoreactivity bands were revealed by chemiluminescence using ECL detection system (Biorad). Chemiluminescence and fluorescence membrane signals were scanned and quantified in an Odyssey LI-COR scanner (Carlo Erba Reagents).