Anti c3ar

Deparaffinized and rehydrated 3 μm thick muscle sections collected before at the indicated time points after induction of normal weight and obese mice were microwaved with CitraPlus solution (Biogenex, San Ramon, California, USA) for antigen retrieval. Sections were then incubated in 3% H₂O₂ for 15 min to prevent endogenous peroxidase activity, whereas unspecific binding sites were subsequently blocked by applying blocking solution (TBS + 0.3% Tween20 + 10% goat serum) at RT for 30 min. Anti-Pax7 (ThermoFisher Scientific, Dreieich, Germany; PA1-24471; 1:750; n = 6), anti-C3ar (Bioss Antibodies; Woburn, MA, USA; bs-2955R; 1:100; n = 3) or anti-C5ar (proteintech®; Manchester, United Kingdom; 21316-1-AP; 1:100; n = 3) were applied and incubated at RT for 2 h. Goat anti-rabbit secondary antibodies (N-histofineH from Nichirei Corporation, Tokyo, Japan for Pax7; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; 111-035-144; 1:50; for C3ar/C5ar) were then added for 1 h at RT and developed by adding the chromogen 3,39-diaminobenzidine (DAB; Agilent, Santa Clara, CA, USA). Sections were counterstained with hematoxylin and briefly immersed into 1% HCl in EtOH to prevent unspecific binding. Sections were then de-hydrated and mounted using Entellan® (Merck, Darmstadt, Germany).