Anti f4 80 antibody

Specimens were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and immunostained for F4/80 or alpha smooth muscle actin (αSMA), as previously reported [15 (link), 16 ]. In brief, heart samples were harvested from PBS- or talc-injected mice and fixed with 4% paraformaldehyde overnight. Tissues were then embedded in paraffin and sectioned into 10 μm slices along the cephalocaudal axis. For avidin-biotin complex (ABC) immunostaining, sections were incubated with anti-F4/80 antibody (1/100 dilution; Clone# A3–1, Abcam, Cambridge, MA) or anti-αSMA antibody (1/100 dilution; Clone# SPM332, Novus Biologicals, Littleton, CO, USA) overnight, followed by horseradish peroxidase-labelled secondary antibody (Nichirei Bioscience, Tokyo, Japan) for 1 h. Visualization of F4/80 and αSMA was achieved via 3,3′-diaminobenzidine tetrahydrochloride (DAB) reaction. Images were taken using a microscope (Olympus, Tokyo, Japan).

The dewaxed tissue sections were alternately placed in citric acid buffer and heated to boiling point for antigen repair for 20 min, and then returned to room temperature. The cells were permeated with PBS which contained 0.3% Triton X-100 for 20 min. These slices were closed with 3% Donkey serum and incubated in an incubator for 30 min. The specific fluorescent antibody was incubated overnight at 4°C and the fluorescent secondary antibody was incubated for 1 h. Primary antibodies anti-F4/80 antibody (1/100, abcam), anti-iNOS antibody (NOS2) (1/100, abcam), and anti-CD206 (MMR) monoclonal antibody (MR6F3)-PE (1/50, invitrogen) were utilized. For anti-F4/80, anti-iNOS and anti-CD206 staining, donkey anti-rabbit (1/500, Alexa 488,abcam), goat anti-rabbit (1/500, Alexa 647,Invitrogen), and donkey anti-goat (Alexa Fluor^®555) secondary antibodies were used. After antibody incubation, anti-fluorescence quenching sealing tablets (including DAPI) were added to seal the tablets. Images were collected and analyzed by laser confocal microscopy system (TCS SP8 X, Leica). Immunofluorescent antibody markers were matched with protocols provided in the relevant literature (Yu et al., 2016 (link)). F4/80⁺CD206⁻iNOS⁺ cells were M1-type macrophages, and F4/80⁺CD206⁺ iNOS^-cells were defined as M2-type macrophages.

Macrophage contents in atherosclerotic lesion were measured using immunohistochemistry staining. Briefly, aortic sinus sections were incubated with 3% H₂O₂ for 10 min and blocked with 3% BSA (Sigma) for 1 h and incubated with anti-F4/80 antibody (1:200, Abcam, Inc., CA, USA; Cat. No.ab-300421) overnight at 4 °C. After incubating with anti-rabbit lgG for 1 h at room temperature, slides were developed with 3,3′-diaminobenzidine (DAB Quanto Kit, TA-060-QHDX, ThermoFisher) and stained with heamatoxylin. Images were recorded using a light microscope.

We detected apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Millipore, MA, USA). Kidney sections were prepared according to the procedure of the Vectastain Elite ABC kit (Vector Laboratory, CA, USA). Anti-CD3 antibody (Santa Cruz Biotechnology, CA, USA) and anti-F4/80 antibody (Abcam, Cambridge, UK) were used and revealing reaction was performed with an AEC Chromogen Kit (Immunotech, Marseille, France). All of these sections were examined in a blinded manner using light microscopy. The number of positive cells was quantified per high power field for each kidney and at least 20 fields were reviewed for each slide.
For immunofluorescence staining, sections were incubated with anti-EP4 antibody (Santa Cruz Biotechnology) and anti-aquaporin-1 (AQP-1) antibody (Millipore) overnight and then visualized with anti-rabbit Alexa Fluor-488 and -594 (Life Technologies, OR, USA). The images were captured by confocal microscopy (LSM5 Live Configuration Variotwo VRGB; Zeiss, Germany).