Rabbit anti mouse cd206

To detect microglial phenotypes, BV-2 microglial cells (2.5 × 10⁴/well) in a 24-well plate were first dispensed on a 12 mm-diameter coverslip to assure about 70% cell density. A mouse anti-mouse ARG1 antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rat anti-mouse CD68 (1:1,000, Abcam, Cambridge, MA, USA) was used to mark M1-like microglial phenotype. A rabbit anti-mouse CD206 or rat anti-mouse CD16 (1:1,000, Abcam) was used to mark M1-like microglial phenotype. Species-specific Alexa Fluor 488 or 555-conjugated anti-rat, anti-mouse, or anti-rabbit secondary antibodies (1:500, CST, Danvers, MA, USA) was used to detect any positive signal, respectively. Thereafter, cells were counterstained with DAPI. The reaction product was absent when the primary antibody was omitted. The number of microglial cells labeled with positive M1-like or M2-like signal as well as the total number of cells was respectively counted in five randomly selected fields under 40× magnification by using a laser confocal microscope (Nikon DS-Ri2, Japan). Then, the percentage of positive cell number relative to the total cells was calculated in different treatment groups.

Tissue sections were immunofluorescently stained with the following primary antibodies: rat anti-mouse Mac2 (1:200; Cedarlane Corp., Burlington, Ontario, Canada), rabbit anti-mouse CD206 (1:300; Abcam, Cambridge, MA, USA), and guinea pig anti-mouse perilipin (1:200; Progen, Heidelberg Germany), After washing, the samples were incubated with donkey anti-rat-555 IgG (1:200; Abcam) and goat anti-rabbit-430 IgG (1:200; Invitrogen, North Ryde, NSW, Australia). Nuclei were stained with DAPI 33342 (Solarbio, D8200, Beijing),and then samples were examined under a light microscope (Nikon Ni-U, Japan). To assess the regeneration of adipose tissue, the preadipocytes^{13 (link)} were counted in 20 randomized fields on each section (200× magnification). To investigate the effect of two injection strategies on the extent of adipose tissue macrophage infiltration, we evaluated the fluorescence staining of M1(red) M2(yellow) macrophages at 100x magnification. A blinded method was adopted in our experiment. The sample for each mark was unknown by the investigator.

Tissue samples were stained with rat anti‐mouse MAC2 (1:200; Cedarlane Corp., Burlington,), rabbit anti‐mouse CD206 (1:300; Abcam,), rat anti‐CD31 (1:200, Abcam), rat anti‐CD34 (1:200, Abcam), rabbit anti‐mouse perilipin (1:400; Progen, Heidelberg, Germany) and chicken anti‐GFP (1:200, Abcam) primary antibodies. After washing, the samples were incubated with donkey anti‐rat Alexa Fluor 555 IgG (Abcam) and goat anti‐rabbit Alexa Fluor 430 IgG (Invitrogen, ) antibodies. Nuclei were stained with DAPI (Sigma, ).