2.8. Caspase-3 Immunohistochemistry and ELISA

Caspase-3 staining utilizing anti-caspase-3 (Rabbit, Abcam, cat. number ab13847) was performed to corroborate apoptosis' involvement in DIMT. This stain was carried out following standard staining procedures, as published previously [2]. In brief, the samples were blocked with NGS and washed with 1x PBS, and the primary antibody, anti-caspase-3 (1 : 50), was diluted in 10% NGS and incubated at 4°C overnight. The slides were washed with 1x PBS, and the secondary antibody, Alexa Fluor 568 goat anti-rabbit, was added and allowed to incubate for one hour at room temperature. The slides were washed one more time and finally were mounted and stained with DAPI.

Caspase-3 activity was analyzed using an ELISA kit from BioVision according to the manufacturer's instructions and as previously reported [17]. Soleus muscle was washed with 1x PBS and homogenized in cell lysis buffer. The sample was then centrifuged to isolate the supernatant. Then, the sample was analyzed to determine protein concentration at 405 nm using a plate reader.