Rabbit anti bdnf

Rat brain tissue or cell samples were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail as previously described. Lysates were centrifuged and collected. Equal amounts of total protein ranging from 15 to 30 μg total protein were separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The following antibodies were used: rabbit anti-cfos (1:1000, Abcam), rabbit anti-CX3CR1 (1:3000, Abcam), goat anti-FKN (1:200, Abcam), rabbit anti-BDNF (1:3000, Abcam), rabbit anti-GAPDH (1:3000, Servicebio), and goat anti-iba1 (1:500, Woko). Blots were developed with an HRP-labelled goat anti-rabbit antibody (1:5000, Servicebio) and an HRP-labelled donkey anti-goat secondary antibody (1:5000, Servicebio). Protein bands were visualized using a chemiluminescence system (ChemiDocTM XRS + , BioRad). Protein expression was semiquantitatively analysed with Image J software.

Animals were sacrificed after deeply anesthetized with isoflurane, total proteins from the lumbar enlargements of spinal cords were extracted by ice-cold Radio Immunoprecipitation Assay (RIPA) lysis. Proteins were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). After blocking with 5% nonfat milk, the samples were incubated with rabbit anti-β-actin (1:3000, Abcam, United States), rabbit anti-GAPDH (1:3000, Merck Millipore, United States), mouse anti-VGluT2 (1:2000, Abcam, United States), goat anti-Iba-1 (1:2000, Wako, Japan), rabbit anti-GFAP (1:2000, Abcam, United States), rabbit anti-BDNF(1:2000, Abcam, United States), or rabbit anti-TrkB (1:2000, Abcam, United States) at 4 °C overnight. The next day, the corresponding rabbit, goat, or mouse Horseradish peroxidase (HRP)-conjugated secondary IgG (1:20000, Jackson ImmunoResearch, United States) was incubated for 1 h at room temperature. The gray value of the protein on the membrane of the corresponding molecular size was detected using western blotting detection kit (WesternBright Sirius, Advansta, United States) and ChemiDoc XRS imaging system (Bio-Rad). Image Lab 3.0 system (Bio-Rad) was utilized to perform standardized analysis on the test results.

Total proteins were extracted from cortical tissues. BCA reagent (Beyotime, Shanghai, China) was used to measure the concentration of extracted proteins. A total of 10 μg protein sample was loaded for polyacrylamide gel electrophoresis (10% gel for TrkB, 12% gel for BDNF), and were transferred to polyvinylidene fluoride membrane (Pall Life Science, Port Washington, NY, USA) in a 200 V electrical field for 30 min. The membrane was first blocked for 1 h using 5% defatted milk powder, and then was incubated with rabbit anti-BDNF (1:2000, Abcam, Cambridge, MA, USA), goat anti-TrkB (1:1000, R&D System, Minneapolis, MN, USA) or rabbit anti-tubulin (1:2000, Abcam) primary antibody at 4°C overnight. Excess antibody was then washed by PBST, followed by incubation in horseradish peroxidase (horseradish peroxidase-conjugated goat anti-rabbit IgG (1:8000, ServiceBio, Wuhan, China) or mouse anti-goat IgG (1:10 000, ServiceBio) for 1 h at room temperature. The membrane was developed in ECL chromogenic substrates, and was imaged by an automatic protein imaging system (Bio-Rad, Hercules, CA, USA). Image J (NIH, Bethesda, MD, USA) software was used to measure optical density values of each band, which was normalized to that of tubulin. Each experiment was performed in triplicates.