Rabbit anti gapdh

Specified brain regions in the ipsilesional brain hemisphere, including the peri-infarct area and hippocampus, were freshly dissected in ice-cold PBS and lysed in RIPA lysis buffer. The homogenates were centrifuged (14000×g, 10 min, 4°C), and proteins were collected from the supernatant. Protein samples from each group were loaded onto SDS-PAGE gels (4%-12%), separated electrophoretically and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4°C with one of the following primary antibodies: rabbit anti-NeuN (1:5000, Abcam), rabbit anti-SYP (1:20000, Abcam), rabbit anti-GAP43 (1:1000, Abcam), or rabbit anti-GAPDH (1:2000, Sangon Biotech, Shanghai, China). After washes with Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature and then washed again. Protein bands were detected with an enhanced chemiluminescence (ECL) kit (CWBIO, Beijing, China). An investigator blinded to the experimental groups quantified the optical density after normalization to GAPDH by using Gel Analysis V 2.02 software 9 (Clinx Science Instruments, Shanghai, China).

By drawing growth curves at different time points, and cultures were adjusted to an OD600 of 1.9 by dilution or concentration. Moreover, surface proteins were extracted with 5 M LiCl from lactobacilli harvested at different time points as previously described [47 (link)]. Immunoblotting analysis was performed as previously described [27 (link)]. The primary antibody was rabbit anti-GAPDH generated in our previous study (Sangon Biotech, Shanghai, China; 1:1000) [27 (link)]. The specific proteins were detected by using an enhanced chemiluminescence kit (Perkin Elmer Life Sciences, Boston, MA, USA). Protein bands were visualized with a chemiluminescent substrate and a gel-imaging system (Tanon Science and Technology, Shanghai, China) and analyzed via the Image Analysis Software (National Institutes of Health, Bethesda, MD, USA).