Once the samples were extracted, they were kept in formol, glutaraldehyde and a third sample was cryopreserved at -80°C for further studying [40]. For histochemical procedures, all muscle specimens were first dissected Nutlin-3 price free of visible connective tissue and fat and embedded in paraffin using conventional methods. Ten-micrometre sections were cut,
varying the inclination of the holder by 5-degree increments until the minimum cross-sectional area was obtained, which was defined as truly transverse. Evaluation of sarcolemmal disruptions Evaluation of sarcolemmal disruption relies on the observation that cellular membrane permeability to albumin is a sign of membrane injury [41]. For assessment and quantification of muscle membrane injury, we chose a method based on light microscopy for identification of fibres that contain albumin by immunohistochemistry. Each sample was processed for immunohistochemical techniques using a polyclonal rabbit anti-human antibody directed against albumin (Code No. A0001; Dako Cytomation, DK-2600 Glostrup, Denmark) as a primary antibody. This immunocomplex was detected using a horseradish peroxidase-labelled goat anti-rabbit secondary antibody (Code No. K4003; EnVision + System-HRP labelled polymer, Dako Co., Carpinteria, CA, USA). The reaction was developed with a chromogen solution with 3.3-diaminobenzidine (Code No. K3468; Liquid DAB + Substrate-Chromogen learn more Solution, Dako Co.,
Carpinteria, CA, USA). The analysis of intracellular albumin was performed by two independent observers using a categorical scale (0–3) with a light microscope (Olympus, Series AX70TF; Olympus many Optical Co., Shinjukuku, Tokyo, Japan) coupled with an image-digitizing camera (View Finder Lite; Version 1.0.143c; Pixera Co., Los Gatos, CA) and a morphometry program (Scion Image, Version Beta 4.0.2; Scion Co., Frederick, MD, USA). Qualification of fibre injury was performed in a four-category finite interval system, the extremes representing either the absence of intracellular albumin (i.e., absence of sarcolemmal injury, degree 0), or presence of intracellular
albumin on the complete cellular area (i.e., severe sarcolemmal injury, degree 3). The two intermediate categories were classified as degree 1 injury (i.e., mild sacolemmal injury or presence of albumin in less than 50% of the fibre area) and degree 2 injury (i.e., moderate sarcolemmal injury or presence of albumin on more than 50% of the fibre area, but not in all of it). Fibre categories were expressed as proportion (%) of total muscle fibres. The mean value of degree 2 and degree 3 obtained by two observers was used for statistical analysis [42, 43]. CD3+ and MPO immunohistochemical staining For the assessment and quantification of CD3+ and MPO intra/interfibrillar infiltrates we chose a method based on light microscopy for identification of CD3+ and MPO by immunohistochemistry. For the immunohistochemical assay we used manual immunostaining.