Sc 65514

Mice were sacrificed by decapitation immediately after FST, the hippocampus was quickly isolated from brain. The hippocampus was homogenized in RIPA buffer containing protease inhibitor (Thermo Scientific, Waltham, MA, USA). The quantified proteins were separated by electrophoresis on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h at room temperature using 4% skimmed milk in Tris-buffer saline and probed overnight at 4 °C with primary antibodies raised against pro-BDNF (brain-derived neurotrophic factor) (1:1000 dilution, sc-65514, Santa Cruz Biotechnology, CA, USA). The membranes were incubated with the horseradish peroxidase-linked secondary antibody for 2 h. Then, immunoreactive proteins were detected by imaging systems; chemiluminescence (LI-COR Biosciences, Lincoln, NE, USA) and determined using the ImageJ software (NIH, Bethesda, MD, USA).

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 total hippocampus protein was extracted by RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice and quantified using a BCA protein assay kit. Each sample (30 µg total protein) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 5% skim milk for 2 h at RT and incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were washed three times with TBST (0.1% Tween-20) and incubated with HRP-labeled secondary antibodies for 2 h at RT. The bands were detected using an enhanced chemiluminescence (ECL) system and analyzed by Image J Software. The primary antibodies used were as follows: iNOS (1:500, sc-7271, Santa Cruz, CA, USA), CD206 (1:500, sc-70586, Santa Cruz, CA, USA), GFAP (1:500, sc-33673, Santa Cruz, CA, USA), C3 (1:500, sc-28294, Santa Cruz, CA, USA), proBDNF (1:500, sc-65514, Santa Cruz, CA, USA), TrKB (1:500, sc-377218, Santa Cruz, CA, USA), BDNF (1:1000, 28205-1-AP, proteintech, Wuhan, China), p75 (1:500, sc-271708, Santa Cruz, CA, USA) and β-actin (1:1000, #4967, CST, Danvers, MA, USA). The secondary antibodies were HRP-conjugated anti-mouse (1:1000, #7076, CST, MA, USA) and HRP-conjugated rabbit (1:1000, #7074, CST, MA, USA).