Sc 547

Western blot analysis was performed as described [83 (link)].
Briefly, after protein extraction from brain tissues, cytosolic lysates were used for the detection of BDNF and NT-3.
After SDS-PAGE, the proteins present in the polyacrylamide gel are transferred to the PVDF membrane. Membranes were incubated at 4 °C overnight with each of the following primary antibodies: anti-BDNF (1:500; sc-20981; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NT-3 (1:500; sc-547; Santa Cruz Biotechnology, Dallas, TX, USA) dissolved in a PMT solution consisting of 1× phosphate buffer saline (PBS), 5% w/v nonfat dried milk powder and 0.1% Tween-20. Thereafter, the membranes were washed and incubated with secondary antibody (1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature.
To confirm that the samples used contained a uniform concentration of protein lysates, they were incubated in the same way, with primary anti-β-actin antibody (1:500; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). Signals were exposed with chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions (Thermo, Waltham, MA, USA). The relative expression of the protein bands was quantified by densitometry and standardized to β-actin levels, as an internal control.

Total protein was extracted from microglia by scraping the cells in SDS/sample buffer. Tissue from cerebellar organotypic slices was directly heated at 100°C in sample buffer (7 min). Samples were loaded and size‐separated by electrophoresis using Criterion TGX Precast 12% gels and transferred to Trans‐Blot Turbo Midi PVDF Transfer Packs (Bio‐Rad, Hercules, USA). Membranes were blocked in 5% skimmed milk and 5% serum in Tris‐buffered saline/0.05% Tween‐20 (TBS‐T) and proteins detected by specific primary antibodies to BDNF (#sc‐547, 1:200; Santa Cruz), to MBP (#SMI‐99P, 1:2,000, Covance), to GAPDH (#MAB374, 1:2,000; Millipore), and to β‐actin (#A2066, 1:1,000; Sigma), followed by secondary peroxidase‐coupled goat anti‐rabbit antibodies (#A6154, 1:2,000; Sigma) or sheep anti‐mouse antibodies (#A6782, 1:2,000; Sigma). After washing, blots were developed using an enhanced chemiluminescence detection kit according to the manufacturer's instructions (SuperSignal West Dura or Femto, Pierce). Images were acquired with a ChemiDoc MP system (Bio‐Rad) and quantified using ImageJ software. Values of BDNF and MBP were normalized to corresponding β‐actin and GAPDH signal, respectively.

Every one-tenth of the continuous sections of each sample was used to perform immunohistochemistry. Briefly, after being washed with PBS, the sections were penetrated by 1% Triton X-100 for 1 h and incubated with blocking buffer at room temperature (RT) for 1 h. Subsequently, the tissues were incubated with primary antibodies overnight at 4°C, fluorescent 488- or 568-conjugated secondary antibodies (1 : 400) for 2 h at RT, and 4′,6-diamidino-2-phenylindole (DAPI, 1 : 2000, Sigma, D9542) for 2 min. Finally, the sections were coverslipped with Vectashield (Vector). The following primary antibodies were used: mouse anti-glial fibrillary acidic protein (GFAP, 1 : 500, Abcam, ab10062), rabbit anti-neuron-specific nuclear protein (NeuN, 1 : 400, Abcam, ab177487), rabbit anti-brain-derived neurotrophic factor (BDNF, 1 : 400, Abcam, ab6201), rabbit anti-nerve growth factor (NGF, 1 : 200, Millipore, 04-1119) or rabbit anti-neurotrophin-3 (NT-3, 1 : 200, Santa Cruz, SC-547) for spinal cord sections, and rabbit anti-growth-associated protein 43 (GAP-43, 1 : 500, Abcam, ab16053) for sciatic nerve sections.