Anti zo1 tight junction

The mouse gastrocnemius muscle was fixed in 4% paraformaldehyde (#158127; Sigma-Aldrich) overnight at 4°C. The tissues were embedded in opti-mum cutting temperature compound (OCT) (#4583; Sakura, Torrance, CA, USA), before incubating frozen sections of 5 μm with 5% goat serum, blocked for 1 h, and immunostaining with Anti-ZO1 tight junction (#61-7300; Invitrogen, Waltham, MA, USA) overnight. The sections were then incubated with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) for 1 h and were nuclear-stained with DAPI (P0131; Beyotime, Shanghai, China). A representative picture was taken with confocal microscopy (LSM710; Carl Zeiss, Oberkochen, Germany) at 400 × magnification.

The gastrocnemius (GC) muscles were excised from mice and immersed in TISSUE TEK O.C.T compound (#4583, Sakura, USA) for cryo-embedding. Five-micrometer cross-sections were cut on slides. The slicing was fixed in 4% paraformaldehyde for 15 min at room temperature and rinsed three times in PBS for 5 min each to remove OCT. Then, the slicing was blocked with 10% bovine serum albumin (BSA) (Sigma, Burlington, MA, United States) for 1 h. Aspirated BSA solution and the sections were incubated with Anti-ZO1 tight junction (#61-7300, Invitrogen) overnight. CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (#SA00013-2, Proteintech) was used as fluorochrome-conjugated secondary antibody, incubated for 1 h at 37°C from light, and nuclear stained with DAPI (#P0131, Beyotime). The representative magnified images were captured with Confocal microscopy (CarlZeiss LSM710; Carl Zeiss) at 20× magnification under the same brightness/contrast setting.