Secondary biotinylated goat anti rat igg

Liver sections were stained with hematoxylin and eosin (H&E). The necrotic area was assessed by analyzing at least 10 randomly selected areas per sample, with computer-assisted image analysis with MetaMorph software (Universal Imaging Corporation, Downingtown, PA). Liver macrophages and neutrophils were detected using primary rat anti-mouse F4/80⁺ mAb (Mac-1, M1/70; BD Biosciences, San Jose, CA) and Ly6G⁺ mAb (BD Biosciences, San Diego, CA), respectively. After incubation with secondary biotinylated goat anti-rat IgG (Vector, Burlingame, CA), followed by treatment with immunoperoxidase (ABC Kit, Vector), the average number of positive cells was quantified by analyzing at least 10 random high-power fields (HPF, original magnification × 400) per animal, with Image-Pro Plus software.

Liver macrophages and neutrophils were detected using primary rat anti-mouse CD11b⁺ mAb (Mac-1, M1/70; BD Biosciences, San Jose, CA) or Ly6G⁺ mAb (BD Biosciences, San Diego, CA). After incubation with secondary biotinylated goat anti-rat IgG (Vector, Burlingame, CA), followed by treatment with immunoperoxidase (ABC Kit, Vector), positive cells were counted blindly in ten HPF/section (×400). For immunofluorescence, frozen liver sections were labeled with primary antibodies ATF3 and CD11b (Santa Cruz Biotechnology, CA), and then incubated with secondary Cy3-conjugated AffiniPure donkey anti-goat IgG antibody (Jackson Immunoresearch, PA). The samples were pre-mounted with VECTASHIELD medium with DAPI.