Rat anti mouse f4 80

Formalin-fixed frozen livers were stained with Sirius Red and anti-α-SMA Ab (Abcam). Immunohistochemistry was performed using DAB staining (Vector), and counterstaining with Hematoxilin. The following antibodies were used for immunostaining: rabbit anti-mouse α-SMA (Abcam, Cambridge, MA), rabbit anti-mouse Desmin (Thermo Fisher Scientific, Fremont, CA), rat anti-mouse F4/80 (eBioscience, San Diego, CA), or rabbit anti-mouse 4-HNE (Alpha Diagnostic Intl Inc., San Antonio, TX) antibodies, following incubation with Alexa Fluor ® secondary antibody. The images were taken using Nikon microscope, and analyzed by Image J.

Snap-frozen whole TA muscle was transversely cryosectioned (8μm), and either stained with hematoxylin and eosin or prepared for immunofluorescence. For immunofluorescence, muscle was fixed with cold acetone and incubated with rat anti-mouse F4/80 (1:200, eBioscience, USA); rat anti-mouse CD11b (1:200, eBioscience, USA); rat anti-mouse CD8α (1:200, eBioscience, USA); rat anti-mouse H-2K^b (1:100, BD, USA); rabbit polyclonal anti-caspase3 (1:500, abcam, USA);rabbit polyclonal anti-dystrophin (1:200, Santa Cruz, USA); or rabbit polyclonal anti-CD3ε (1:200, Abcam, USA). Rhodamine-conjugated goat anti-rat IgG (1:200, Santa Cruz, USA), FITC-conjugated goat anti-rabbit IgG (1:200, Santa Cruz, USA) or Alexa 568-conjugated rat anti-mouse IgG (1:1000, Invitrogen, USA) were used as secondary antibodies. Nuclei were counterstained with DAPI. Slides were viewed with an Olympus BX51 fluorescence microscope (Olympus, Japan). For results analysis, the Image J software was performed to quantify the intensity of staining. The integrated optical density (IOD) and area of interest (AOI) of all the positive staining (intense red staining) were measured, respectively. The mean density (IOD/AOI) was then calculated. Cell apoptosis was determined by counting the number of CD11b⁺caspase-3⁺ cells and expressed as a percentage of total CD11b⁺cells counted.