Western Blot Analysis of Brain BDNF

For Western blot analysis, brain cortexes were quickly collected and frozen in dry ice. Tissue samples were homogenized in RIPA buffer and quantified using the Bradford method, as previously described [14 (link)]. Equivalent amounts (50 μg) of protein were subjected to electrophoresis on Bolt^TM 4–12% Bis-Tris Plus gel (Life Technologies Corporation, Carlsbad, CA, USA, No. 04127) and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Science, Piscataway, NJ, USA, No. 10600003). The membrane was cut above the 25 KDa and the 50 KDa band of the marker. The two lower parts of the membrane were separately incubated with an anti-BDNF and an anti-β-actin antibody, respectively, to avoid membrane stripping and reprobing (Figure S1). The primary and secondary antibodies used are listed in Table S2. Densitometric analysis of digitized Western blot images was performed using ChemiDoc XRS Imaging Systems and the Image Lab^TM Software version 5.2 (Bio-Rad, Hercules, CA, USA). This software creates automatic highlights of any saturated pixels in the Western blot images. Images acquired with exposition times that generated protein signals out of a linear range were not considered for quantification.