Rabbit anti zo 1 polyclonal antibody

Ipsilateral hemispheres protein homogenate was extracted from mouse brain 3 days after ICH. Western blotting was performed as we previously described [21 (link)]. Proteins were electrophoresed and transferred onto a PVDF membrane (Merck KGaA, Darmstadt, Germany). After being blocked, membranes were incubated overnight at 4 °C with mouse anti-EMMPRIN monoclonal antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-MMP-9 polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-ZO-1 polyclonal antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-occludin polyclonal antibody (1:1000, Abcam, Cambridge, MA, USA), and GADPH antibody (1:5000, Proteintech, Wuhan, China). Afterward, membranes were incubated for 1 h at room temperature with the HRP-conjugated anti-rabbit IgG antibody (Servicebio, Wuhan, China) or the anti-mouse IgG antibody (Servicebio, Wuhan, China). Finally, the protein-specific signals were detected using a BIO-RAD ChemiDoc™ MP Imaging System.

Polarized monolayers of MDCK-mock and MDCK-fpIgR cells were grown on 6.5-mm filters for 3–4 days. The filters were washed thrice in ice-cold PBS (5 min each time) and then incubated on both sides of the chamber with 4% paraformaldehyde for 20 min. Cells were washed thrice with PBS and blocked in PBS containing 5% BSA for 1 h at 37°C and washed as described above. Following incubation overnight at 4°C with a rabbit anti-ZO-1 polyclonal antibody (Proteintech, Chicago, IL, USA) diluted 1:400 in PBS, cells were washed thrice with PBST for 15 min and incubated with Cy3-labeled anti-rabbit IgG antibody for 45 min at 37°C. After DAPI staining for intact nuclei, the polyester membrane was cut down with a scalpel and mounted on glass slides using the antifade mounting medium before observation by CLSM.

For Western blot analysis, the rat bladder samples were processed according to the standard protocol (Beyotime, Shanghai, China). Total protein was extracted and quantified using the bicinchoninic acid (BCA) protein assay method. Approximately 40 μg of protein from each sample was separated on 8%, 10%, or 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with defatted milk and incubated overnight at +4°C with specific primary antibodies, including rabbit anti-E-Cadherin polyclonal antibody (1:5,000, Proteintech, United States), rabbit anti-ZO-1 polyclonal antibody (1:5,000, Proteintech, United States), rabbit anti-SNAP-23 polyclonal antibody (1:2000, Abcam, United States), mouse anti-cleaved and uncleaved SNAP-25 monoclonal antibody (1:100, GeneTex, United States), and rabbit anti-GAPDH polyclonal antibody (1:5,000, Proteintech, United States). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000, Proteintech, United States) at room temperature for 1 h. Protein bands were visualized and analyzed using the Bio-Rad ChemiDoc Imagers System.