Rat anti mouse cd206

Balb/c mice were orthotopically injected with 1 million of 4T1 cells in the mammary gland and after 3 days FAM-LinTT1-PS (1mg of polymer, 100μL) was intravenously injected. After 24 h, the animals were sacrificed and the tumor and organs were excised, fixed in 4% of paraformaldehyde, cryoprotected with 15% and 30% sucrose, frozen down with liquid nitrogen, and cryosectioned at 10 μm. Tissue sections were permeabilized using PBS 10 mM containing 0.2% Triton-X for 10 min, and blocked in PBS 10mM containing 0.05% Tween-20, 5% FBS, 5% BSA, and 5% goat serum (GE Healthcare, UK) for 1 h. The sections were immunostained at dilution 1/100 with anti-fluorescein rabbit IgG fraction (Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31, biotin rat anti-mouse CD11b, (BD Biosciences, CA, USA), rat anti-mouse CD68, rat anti-mouse CD206 (Bio-Rad, USA), and anti-p32 rabbit polyclonal antibody (Millipore, Germany) as primary antibodies. As secondary antibodies, Alexa 488-conjugated goat anti-rabbit IgG and Alexa 647-conjugated goat anti-rat IgG (1/500, Invitrogen, Thermo Fisher Scientific, MA, USA) were used. The sections were counterstained with DAPI and examined by fluorescence confocal microscopy using Olympus FV1200MPE instrument. The images were processed and analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany) and the Image J software.

The mouse aortas were harvested on days 7 or 14, embedded in OCT and sectioned. Cross sections of aortic tissues (9 µm) were fixed in 4% paraformaldehyde, blocked in 8% BSA in PBS and incubated with the following primary antibodies: rat anti-mouse MOMA-2 (1:200 dilution; Cat# ab33451, Abcam, Cambridge, United Kingdom), biotinylated-IL-12p40 (1:100 dilution; Cat# 505302, Biolegend, San Diego, CA), rat anti-mouse CD206 (1:200 dilution; Cat: MCA2235, Bio-Rad [Formerly AbD Serotec], Hercules, CA), or rabbit anti-mouse CD8a (1:200 dilution; Cat# bs-0648R, Bioss antibodies, Woburn, Massachusetts) followed by the appropriate rhodamine red- or FITC-conjugated secondary antibody (1:100–1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) or Streptavidin Alexa Fluor 488 conjugate (1:400 dilution; Cat# S-11223, Molecular Probes, Eugene, OR). Nuclei were counterstained with DAPI. Images were acquired on a Leica DM 2000 microscope fitted with a Leica DMC 4500 color camera and analyzed with Leica Application Suite (LAS) X software. Single- or double-color images were loaded into ImageJ and cell enumeration was performed in a blinded fashion. Data were obtained from 3–4 non-overlapping fields per aortic cross-section, 6–9 sections per aorta, n = 6–8 aortas per time point.