Goat anti rat igg fitc

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.

Immunohistochemical detection of F4/80 and myeloperoxidase (MPO) was performed as previously described32 (link). The number of F4/80- or MPO-positive cells were quantified from 5–7 fields from 3–10 mice/group. F4/80 labeling was carried out in frozen liver sections fixed in 4% paraformaldehyde using a rat monoclonal anti-F4/80 antibody (1:20, Serotec). Labeling was achieved using a goat anti-rat IgG FITC (1:50, Serotec). No staining was observed when omitting the primary antibody. The number of F4/80-positive cells was quantified from 3–6 fields/animal and from 2–4 mice/group.