Horseradish peroxidase conjugated goat anti rabbit igg

On day 70, samples from the cerebral cortex were collected on ice and stored at –80°C in a freezer. The samples were lysed with lysis solution mixed with 1% phenylmethylsulfonyl fluoride to isolate the protein, and the protein concentration of each sample was determined. The electrophoresis device was assembled, and the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Cat# FFP24; Beyotime). After the membrane was blocked with 1% bovine serum albumin (Cat# ST023-50g; Beyotime), it was incubated overnight with the following primary antibodies: rabbit anti-rat GFAP (1:500), mouse anti-rat DCX (1:500), mouse anti-rat NeuN (1:1000), and mouse anti-rat NF200 (1:200). After washing with Tris-buffered saline with Tween-20, the corresponding secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Cat# WLA023; Wanleibio, Shenyang, China) and horseradish peroxidase-conjugated rabbit anti-mouse IgG (1:5000; Cat# WLA024; Wanleibio)) were added and incubated for 45 minutes at 37°C. The electrochemiluminescence reagent (Cat# P0018S; Beyotime) was added and exposed in the darkroom. The data were analyzed with Gel-Pro-Analyzer (Cat# 9413B; Beijingliuyi, Beijing, China).

The cells in different groups were harvested and lyzed in RIPA lysis buffer, followed by centrifuging at 4°C 12,000 g for 10 min. The concentrations of total proteins in individual lysate samples were measured using the BCA protein concentration assay kit (Wanleibio, Shenyang, China). Individual lysates (40 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide-gel-electrophoresis on 10% gels and transferred onto polyvinylidene difluoride membrane (Millipore, USA). The membranes were treated with 5% fat-free dry milk in TBST and incubated with rabbit anti-CCR7 (Bioyanbio, Zhenjing, China) and anti-β-actin antibodies (Wanleibio, Shenyang, China) at 4°C overnight. The membranes were treated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Wanleibio, Shenyang, China) and visualized by the enhanced chemiluminescent reagent, followed by imaging in the Gel Imaging System (LiuYi, Beijing, China). The relative levels of CCR7 to β-actin protein expression were analyzed using Gel-Pro-Analyzer software 6.0 (Media Cybernetics US).