Muscle weight was analyzed to assess the effects of denervation, and the whole biceps was removed for histological analysis. Both analyses were performed on the treated and contralateral biceps to serve as a control. After completion of the maximum muscle force and the MUNE protocol, biceps muscles were carefully dissected, tendons were removed, and weight was assessed using a microscale. Afterward, muscles were embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek, CA, USA), frozen in liquid nitrogen–cooled isopentane, and stored at −80°C for a minimum of 24 hours. The effect of the nerve transfer on fiber populations was analyzed using an immunohistochemistry protocol against MHC subtypes (37, 38). Modifications were made for better contrast and automated analyses as previously described (39, 40). Full cross-sectional samples with a width of 20 μm were stained with a primary antibody cocktail of phosphate-buffered saline (PBS) with 10% goat serum containing MHC-I (BA-F8; 1:50), MHC-IIa (SC-71; 1:600), and MHC-IIb (BF-F3; 1:100) antibodies for 60 min [Developmental Studies Hybridoma Bank (DSHB), Iowa, USA]. PBS containing 10% goat serum was used as a blocking buffer. PBS with 10% goat serum was mixed with secondary antibodies against Alexa Fluor 633 immunoglobulin G2b (IgG2b) (1:250), Alexa Fluor 488 IgG1 (1:250), and Alexa Fluor 555 IgM (1:250) and applied for 60 min (Life Technologies, CA, USA). Per animal, the entire muscle’s cross section was analyzed using an automated analyses protocol as previously described (39, 40).

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