Soon after sacrifice, 3 half-hearts and 3 quadriceps were harvested from controls and from SOD1G93A mice, embedded in OCT and snap-frozen in precooled isopentane. Six-micrometer-thick serial cryostat sections were obtained.
For the immunofluorescence analysis, immediately after cutting, the sections were fixed for 10 min in cold acetone, followed by incubation with MitoTracker® Red (Molecular Probes), at 10 nM for 20 min at 37 °C. For reactive oxygen species (ROS) staining, serial sections from the same samples were fixed for 10 min in acetone, followed by incubation with 10 μM 2′,7′-dichlorofluorescein diacetate (H2DCFDA; Molecular Probes) for 30 min and a wash in PBS.
For immunohistochemical staining of skeletal muscles infiltrated macrophages the following antibodies were used: rat anti-mouse CD206 (AbD Serotec, 1:200, 5 μg/ml) to detect macrophages M2, rat anti-mouse CD86 clone PO.3 (Millipore, 1:100, 5 μg/ml) to detect macrophages M1, and rat anti-mouse CD11b (Novus Biologicals, 1:200, 5 μg/ml).
For the immunofluorescence analysis, immediately after cutting, the sections were fixed for 10 min in cold acetone, followed by incubation with MitoTracker® Red (Molecular Probes), at 10 nM for 20 min at 37 °C. For reactive oxygen species (ROS) staining, serial sections from the same samples were fixed for 10 min in acetone, followed by incubation with 10 μM 2′,7′-dichlorofluorescein diacetate (H2DCFDA; Molecular Probes) for 30 min and a wash in PBS.
For immunohistochemical staining of skeletal muscles infiltrated macrophages the following antibodies were used: rat anti-mouse CD206 (AbD Serotec, 1:200, 5 μg/ml) to detect macrophages M2, rat anti-mouse CD86 clone PO.3 (Millipore, 1:100, 5 μg/ml) to detect macrophages M1, and rat anti-mouse CD11b (Novus Biologicals, 1:200, 5 μg/ml).