Bx61 fully motorized fluorescence microscope

The rats of each group (n = 5) were euthanized with an anesthetic overdose (ketamine 200 mg/kg and xylazine 50 mg/kg, i.p. injection). Gastrocnemius muscle samples were cryo-fixed and stored at −80 °C. Then, longitudinal sections of the MTJ region were acquired (Cryostat HM505 E, MICROM^TM). The samples were washed thrice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and permeabilized with Triton X-100 0.1% for 20 min. The treated samples were then washed again with PBS three times.
For MTJ identification and nuclear analysis (sections of 100-µm thickness), immunostaining was performed using Alexa Fluor™ 488 Phalloidin diluted in PBS (1:600, Invitrogen, A12379) for 30 min for the identification of actin filaments (F-actin), and nucleus staining was performed with 4′,6-diamidino-2-phenylindole (Molecular Probes, Eugene, P36935). Images for histological analysis were obtained using a confocal laser scanning microscope (Leica^TM TCSSP5) and analyzed with a Olympus BX61 Fully Motorized Fluorescence Microscope (Shinjuku, Japan) (Figure 5). We measured the nuclear density in the MTJ region (n = 14 images) in an area of 91.3 mm² (1000× magnification).

For telocyte identification, 10-µm-thick sections were collected, immunostained with primary antibody (CD34, 1:1000, IgG polyclonal, Invitrogen, PA5-85917), and diluted in PBS with 1% BSA. After two washes in PBS, the stained slides were incubated with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (1:1000, IgG, Invitrogen, A-11012) and diluted in PBS with 1% BSA for 60 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, P36935). The histological sections were analyzed with a Olympus BX61 Fully Motorized Fluorescence Microscope (Shinjuku, Japan) at a magnification of 200×. We obtained a differential interference contrast image to visualize the muscle-tendon interface.