Rat anti mouse mac 2

In this study, we utilized male mice for the hematopoietic system reconstitution and female mice for conditional KO. Epidermal fat pads were collected from wild‐type and Mafb^−/− mice, and inguinal and ovary fat pads were collected from Mafb^f/f and Mafb^f/f::Tie2‐Cre mice. The collected fat pads were either fixed in 4% paraformaldehyde solution and embedded in optimum cutting temperature compound or fixed in neutral‐buffered formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (HE), and adipocyte size was measured using imagej analysis software (NIH, Bethesda, MD, USA). The frozen sections were incubated with rabbit anti‐GFP (MBL, Woburn, MA, USA), rabbit anti‐(mouse MafB) (Bethyl, Montgomery, TX, USA) and rat anti‐(mouse Mac‐2) (Cedarlane, Burlington, NC, USA). Alexa Fluor 488‐conjugated goat anti‐(rabbit IgG) (Molecular Probes, Eugene, OR, USA) and Cy3‐conjugated donkey anti‐(rat IgG) (Jackson ImmunoResearch, West Grove, PA, USA) were used as secondary antibodies. Hoechst 33342 (Thermofisher, Rockford, IL, USA) was used to stain nuclei.

Sample sections were stained with the following primary antibodies: rat anti-mouse Mac2 (1:200; Cedarlane Corp., Burlington, ON, Canada), rabbit anti-mouse CD206 (1:300; Abcam, Cambridge, MA, United States), goat anti-mouse perilipin-1 (1:200; Abcam), rat anti-mouse CD31 (1:200; Invitrogen, North Ryde, NSW, Australia), and rabbit anti-human alpha-smooth muscle actin (α-SMA) (1:200; Abcam). After washing, the samples were incubated with donkey anti-rat-555 immunoglobulin G (1:200; Abcam), donkey anti-goat-594 immunoglobulin G (1:200; Abcam) and donkey anti-rabbit-488 immunoglobulin G (1:200; Abcam) secondary antibodies. Nuclei were stained with DAPI (Sigma). The samples were examined under a TCS SP2 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany). Leica LAS AF software was used for images analysis.