Rat anti mouse f4 80 primary antibody

To verify macrophage depletion after the CCL treatment, we performed immunohistochemical staining of the visceral white adipose tissue and liver by using an antibody against F4/80 protein, a specific marker for macrophages. Animals were sacrificed using Isoflurane overdose and transcardially perfused with PBS followed by 4% formaldehyde. Formalin-fixed paraffin-embedded visceral adipose tissues were sectioned (5 μm thick), deparaffinized in xylene, and rehydrated prior to antigen unmasking in proteinase K buffer (Sigma-Aldrich). Sections were blocked with 3% hydrogen peroxide and 5% goat serum and then incubated with rat anti-mouse F4/80 primary antibody (1:200; AbD Serotec, Raleigh, NC) overnight. Sections were then incubated with biotinylated goat anti-rat secondary antibody (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 hours followed by incubation in Avidin-Biotin Complex and developed using 3,3’-diaminobenzidine as substrate (Vector Laboratories, Burlingame, CA). All incubations took place in a humidity chamber at room temperature. Liver sections were counterstained with cresyl violet. Sections were examined using a Leica DM 2000 LED light microscope and images were captured using a Leica ICC50 W Microscope Camera and Leica Imaging Software. F4/80 positive cells were counted on 10 high power (20x) fields per slide (n = 3/condition).