Rat anti f4 80 antibody

Three days after laser injury, the RPE-choroid complex was incubated with isolectin B4-Alexa 488 and rat anti-F4/80 antibody (1:100; Serotec, Oxford, UK). Alexa 546 goat anti-rat secondary antibody (1:200; Thermo Fisher Scientific) was then applied. The isolectin B4-stained area of CNV and F4/80-positive macrophages were quantified, and the area-adjusted number of macrophages was calculated.

Maraviroc was obtained from Sigma-Aldrich (St. Louis, MO, USA) (for in vitro experiments) or GlaxoSmithKline (Brentford, UK) (for in vivo experiments). Mouse CCL3 was obtained from Peprotech (Rocky Hill, NJ, USA). The following antibodies were used as the primary antibodies for immunohistochemical analyses: goat anti-CCR5 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rat anti-Ly6G antibody (BD Biosciences, San Jose, CA, USA), rat anti-F4/80 antibody (Serotec, Kidlington, UK), mouse anti-α-SMA antibody (Dako, Glostrup, Denmark), rabbit anti-type I collagen antibody, rabbit anti-CD31 antibody and rabbit anti-EGF antibody (Abcam, Cambridge, UK). The following rat anti-mouse antibodies were used as the primary antibodies for the flow cytometric analysis: anti-CD11b antibody (BD Biosciences), anti-CD25 antibody (BioLegend, San Diego, CA, USA), anti-CD45 antibody (eBioscience, San Diego, CA, USA), anti-F4/80 antibody (eBioscience), anti-Foxp3 antibody (eBioscience), anti-Ly6G antibody (Gr-1) (Tonbo Biosciences, San Diego, CA, USA), anti-MIP-1α antibody (R&D Systems, Minneapolis, MN, USA), anti-CCR5 antibody (BioLegend), and isotype-matched control IgGs for individual rat antibodies (BD Biosciences).

Cells were fixed in methanol, followed by incubation with a blocking buffer containing 1% BSA and 0.2% Triton X-100 and with a rabbit polyclonal anti-CB2 (1:200, Cayman), a rat anti-F4/80 antibody (1:20, Serotec), a mouse anti-LC3B antibody (1:200, Nano Tools), a guinea pig polyclonal anti-SQSTM1/p62 antibody (1:100, ProGen) or a rabbit anti-HO-1 antibody (1:300) and the appropriate secondary antibodies (goat anti-rabbit IgG Alexa 555 (1:1,000; Invitrogen), goat anti-rat IgG FITC (1:50, Serotec), goat anti-mouse IgG Alexa 555 (1:1,000; Invitrogen), goat anti-Guinea pig IgG Alexa 555 (1:1,000; Invitrogen)) as previously described19 (link). Nuclear staining was performed using Prolong Gold antifade reagent with DAPI (Invitrogen). Fluorescence was imaged on a Zeiss LSM-510 multitracking laser scanning confocal microscope with a Helium/Neon laser at 543 nm and using AxioVision software (Carl Zeiss). No staining was observed when omitting the primary antibody. F4/80- and HO-1-positive cells from 10 fields/conditions were quantified. Results are expressed as percent of HO-1-positive cells per field. The number of SQSTM1/p62 or LC3-positive dots per F4/80-positive cell was quantified from 3–10 fields/condition.