Cd68 antibody

Morphometry of individual fat cells was assessed using digital image analyses as described previously [22 ]. In short: for each subject, the adipocyte cell diameter of all fat cells in five to ten microscopic fields of view were counted and measured. For detection of crown-like structures, a CD68 antibody (AbD Serotec, Oxford, UK) was used in human samples. In mouse samples, an antibody against F4/80⁺ (AbD Serotec, Oxford, UK) was used. Visualization of the complex was done using 3,3’-diaminobenzidene for 5 min. Negative controls were used by omitting the primary antibody.

Gastrocnemius were excised, rinsed in PBS, and frozen in liquid nitrogen. Tissues were cut using a cryostat into 7-μm-thick sections.
Macrophages and CD3-positive cells were visualized after CD68 antibody (1/250, Biorad) or CD3 staining (1/250, DAKO) followed by staining with a donkey 488-AF–anti-rat or anti-rabbit IgG (1/250, Jackson ImmunoResearch) respectively. CD68- and CD3-positive cells were counted in randomly chosen fields with the use of Image J software (NIH). Sections were stained with hematoxylin and eosin (H&E) to analyze muscle structure.

Immunohistochemistry was performed on 12-μm sections of aortic roots freshly embedded in OCT. The slides were fixed in ice-cold acetone for 15 min and permeabilized with PBS + 0.1% Triton X-100 (PBST) for 15 min and blocked with PBST containing 5% BSA (MilliporeSigma, St. Louis, MO) for 1 hour at room temperature. The sections were then incubated with antibodies against CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA), interleukin 6 (IL-6, 1:100; Bio-Rad Laboratories, Hercules, CA), αSMA antibody (1:100; Abcam, Cambridge, United Kingdom), or tumor necrosis factor α (TNFα; 1:100; Abcam, Cambridge, United Kingdom) at 4°C overnight. The slides were rinsed with PBS and incubated with corresponding secondary antibodies (1:500; Life Technologies, Carlsbad, CA). The nuclei were stained by mounting the slides with 4’, 6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories, Burlingame, CA). Images were acquired under a Nikon fluorescence microscope (Nikon, Melville, NY). For collagen staining, Masson’s Trichrome staining was performed following a previously published procedure [58 (link)].

Brains were cut into 25 um thick sections using a microtome (Leica SM2010R, Microtome and Microscope, Leica Microsystems, Wetzlar, Germany) and stored at −20°C in an anti-freeze solution. Brain slices were stained for IIba1 antibody (1:1000, Nordic Biolabs, Wako Chemicals, Täby, Sweden, Cat. #019-19741), CD68 antibody (1:500, Bio-Rad, Hercules, CA, United States, Cat. #MCA1957), TH antibody (1:2000, Chemicon-Millipore, Burlington, MA, United States, Cat. #AB152 and #MAB358), Gephyrin antibody (1:250, Synaptic Systems, Göttingen, Germany, Cat. #147011C3), GFAP antibody (1:500, Dako, Santa Clara, CA, United States, Cat. #Z0334), in PBS with the serum (5–10%) from the animal species of the secondary antibodies and Triton-X 100. The images were acquired using Olympus Virtual Stage 120 with extended focus imaging setting (EFI) for scan images (20x and 10x magnifications), Olympus BX53 microscopes, Shinjuku, Tokyo, Japan (20x and 60x magnification), and LEICA Stellaris 8 Dive for confocal images (40x and 63x magnification). EFI setting consists of five optical sections with a 5 um increment between two consecutive optical sections. Z-stack confocal images consist of 30 optical sections, with 0.5 um increment between two consecutive optical sections, and their 3-dimensional projection was used for analyses.

The same procedure for cryosections was employed in additional hearts for immunofluorescence staining. The cryosections were blocked with 10% bovine serum albumin (BSA) and then incubated for 3 h at 22 °C or overnight at 4 °C with the following primary antibodies: CD68 antibody (1:200; Bio-Rad, Hercules, CA, USA), purified anti-mouse Ly-6G antibody (1:50, Biolegend, San Diego, CA, USA), anti-TNF-α antibody (1:50, Abcam, Waltham, MA, USA), biotinylated nitrotyrosine (3-NT) (1:100; Cayman Chemical, Ann Arbor, MI, USA), anti-DNA/RNA damage antibody, epitope 8-oxo-7,8-dihydro-2′-deoxyguanosine (1:50, Abcam), and anti-iNOS (1:500, Thermo Fisher, Waltham, MA, USA). The sections were washed and incubated with fluorescent-labeled secondary antibody Alexa Fluor-conjugated (Invitrogen, Walthan, MA, USA). The nuclei were counterstained with DAPI for 10 min. The sections were mounted with Vectashield medium, microscopic images of the aortic lesions (objective lenses 10×) were digitalized, and morphometric measurements were calculated as described for Oil red O. ImageJ software, version 1.51s (U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij) was used for all the quantifications.