Horseradish peroxidase hrp conjugated secondary igg

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.

The rats were deeply anesthetized and sacrificed by decapitation after the behavioral tests. The L4-6 spinal cord tissues were quickly dissected and homogenized in a cold RIPA lysis buffer and then centrifuged for 5 min at 14,000× g at 4 °C. The supernatant of the sample was collected and the protein concentration was quantified. Following the addition of a 5 × sample buffer, the samples of the spinal cord were heat-denatured at 100 °C for 5 min. Fifty micrograms of protein per sample were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were then blocked with 5% non-fat milk for 2 h at 24 °C. Thereafter, the membranes were incubated with a specific primary antibody for rabbit anti-GDNF (1:400, Abcam), mouse anti-MOR (1:600, Abcam), and rabbit anti-GAPDH overnight at 4 °C. The next day, the membranes were incubated with the corresponding rabbit, or mouse Horseradish Peroxidase (HRP)-conjugated secondary IgG (1:20,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 24 °C. Reactive bands were visualized with the enhanced chemoluminescent detection method with a digital camera system (cool snap HQ2; Photometrics^®, Tuscon, AZ, USA) and were semi-quantitatively analyzed with a relative density value similar to that for the GAPDH.