Rabbit anti mlck

Tissue or cell samples were washed 3 times with PBS and then treated with 0.5% Triton X-100 in PBS for 30 min before being fixed in 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h. Next, the samples were incubated with the primary antibodies at 4°C for 16–20 h. After being rinsed with PBS, the samples were incubated with the secondary antibodies at 37°C for 1 h. Finally, the samples were mounted using Fluoroshield™ (GeneTex Inc.) containing DAPI, and examined using fluorescence microscopy (Zeiss Apotome 2, JPN). Quantitative analysis was performed using ImageJ software.
We used the following primary antibodies and secondary antibodies at the indicated dilutions: goat anti-CD31 Alexa Fluor 488-conjugated antibody (1:400, R&D, Minneapolis, MN, USA), rabbit anti-Claudin-5 (1:100, Abcam, Cambridge, MA, USA), rabbit anti-Occludin (1:100, CST, Danvers, MA, USA), rabbit anti- zonula occludens (ZO)-1 (1:200, Invitrogen, Carlsbad, CA, USA), rabbit anti-MLCK (1:200, Abcam, Cambridge, MA, USA), rabbit anti-p-MLC (1:50, CST, Danvers, MA, USA), and anti-rabbit Alexa Fluor 488 or Alexa Fluor 594 (1:400, Abcam, Cambridge, MA, USA).

An equal amount of protein exact (20-40 μg) was electrophoresed on a 10% reducing polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. Immunoblots were blocked with 3% bovine serum albumin (BSA) in TBS for 70 min at room temperature and incubated overnight at 4 °C with specific primary antibodies including rabbit anti-occludin (1:1000; Proteintech), rabbit anti-ZO-1 (1:1000; Proteintech), rabbit anti-NF-κB p65 (1:5000; Abcam), rabbit anti-MLCK (1:5000; Abcam), phospho-MLC2 (1:1000; Cell Signaling Technology, Danvers, MA, United States), and phospho-IκBα (1:1000; Cell Signaling Technology) in TBS and 0.05% Tween-20 containing 1% BSA.
Blots were washed and then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 120 min at room temperature. The bands were detected by enhanced chemiluminescence and quantified (relative to β-actin expression) using Scion Image 4.03 analysis software.