Dp72 image system

Muscle samples for histochemistry were taken at the same location. After each tissue was fixed and slides prepared, haematoxylin and eosin staining was performed by a standard method. Slides were viewed by Olympus DP72 image system. For each sample, five view fields (areas) were randomly selected and then analyzed using IPP 6.0 software. The cross-sectional areas of 400–500 myofibers were measured, and average area of each myofiber, total number of myofibers and myofiber density were determined.

Cells were fixed with 1% paraformaldehyde for 10 min at room temperature. The cells were incubated with primary antibodies against anti-Claudin 5 (Cat#35-2500, Thermo-Fisher Scientific) and Occludin (Cat#71-150, Thermo-Fisher Scientific). After overnight incubation at 4 °C, the cells were incubated with fluorescein-labeled secondary antibodies. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Micrographs of all immunostains were acquired by an Olympus BX51 Fluorescence Microscope and an Olympus DP72 image system. Quantification of fluorescence intensity at the cell boundary was performed with Image J (NIH).