Ab108319

BDNF results in multiple bands when separated into its cleavage products. Analysis of specific bands may provide different results and conclusions. To test this, samples from rathlete and sedentary groups were analyzed with three different BDNF antibodies, from three different companies (Novus Cat# NB100-98682, RRID:AB_1290643, Abcam Cat# ab108319, RRID:AB_10862052, Santa Cruz Biotechnology Cat# sc-546, RRID:AB_630940), targeting three different bands in a Western blot (Supplementary Table 1). Included in this is the Novus BDNF antibody used in the main analysis and described in the previous section.

Forty micrograms of total protein lysate was separated on a 15% SDS protein gel with a Kaleidoscope-prestained protein standard (Bio-Rad). Blots were blocked in 5% non-fat dry milk and incubated in primary antibody (mature BDNF, Abcam, Catalog #ab108319 used at 1:5000 and secondary at 1:5000; proBDNF, Santa-Cruz Biotechnology, Catalog #sc-65514 used at 1:500 and secondary at 1:5000) for 24 hours at 4°degrees celsius. Blots were incubated in horseradish peroxidase-linked IgG-conjugated secondary antibody for 1 hour. Protein bands were visualized by chemiluminescence solution (Western Lightening, Perkin Elmer Life Sciences). Band at 15kDa was quantitated for mature BDNF, and band at 28kDa was quantitated for proBDNF. GAPDH (37 kDa; Abcam, Catalog# Ab22555 used at 1:20000 and secondary at 1:30000) was used as a loading control.

The cells grown on slides were fixed with 4% paraformaldehyde for 15 min and blocked with 1× phosphate buffer saline supplemented with 3 mg/mL bovine serum albumin, 100 mM glycine, and 0.25% Triton X-100 for 30 min. Subsequently, the slides were incubated with primary antibody rabbit anti-BDNF (1:500, ab108319), rabbit anti-light chain 3II (LC3II) (1:1000, ab48394), mouse anti-Tau (Tau-13, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-BRUCE (5 µg/mL, ab19609) at 4 °C, followed by culture with the combination of the fluorophore and Alexa Fluor^® 647 secondary antibody (1:200, ab150075) at room temperature for 1 h. All the antibodies used above were provided by Abcam except Tau. Then, the cell slides were added with 4′ 6-diamidino-2-phenylindole for nucleus staining, then immersed in distilled water, dried and observed under a fluorescence microscope (Zeiss, Thornwood, NY) or FV-1000 confocal microscope.
For morphological analysis, the cells were fixed with 4% paraformaldehyde for 15 min and incubated with tau antibody (1:200, ab64193, Abcam) for 1 h, followed by another incubation with fluorophore combined with Alexa Fluor^® 647 secondary antibody (1:200, ab150075) for 1 h. The cells were then observed under the fluorescence microscope, and the length of the main axon in each cell was measured by five independent experiments.