Immunohistochemical Analysis of Myostatin and NF-κB

Immunohistochemistry for myostatin and NF-κB was performed according to a previously described protocol [26 (link)-29 ]. In brief, 4 μm thick sections of paraffin-embedded gastrocnemius skeletal muscle were prepared, deparaffinized, and rehydrated. Next, 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Phosphate-buffered saline (PBS, pH 7.2) was used to wash the sections before and after incubating some of them with myostatin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubating others with anti-NF-κB antibody (Santa Cruz Biotechnology), using the dilutions suggested by the manufacturers. The sections were then incubated with a biotinylated secondary antibody (LSAB kit, Dako, Carpinteria, CA, USA) and washed with PBS before being successively incubated with streptavidin horseradish peroxidase (LSAB kit, Dako). They were then rinsed with PBS and treated with 3,3′-diaminobenzidine substrate (Santa Cruz Biotechnology) until the desired color intensity was detected. Finally, the sections were washed with tap water to stop the further reaction. Hematoxylin (Santa Cruz Biotechnology) was used to counterstain the sections. Negative control slides were processed in the absence of a primary antibody. The sections were viewed at 40x under a light microscope (BEL Engineering Company, Italy).