Goat anti rat af488

Immunohistochemistry (IHC) was performed on mouse tissue that was formalin fixed and paraffin embedded (FFPE). Specimen slides were deparaffinized, rehydrated and then antigen retrieval was performed. The slides were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-mouse CD127, goat anti-mouse IL-22, and a rat anti-mouse lineage cocktail containing CD3, CD4, CD11b, CD19 and F4/80. After the slides were washed, donkey anti-goat AF555, goat anti-rabbit AF647, and goat anti-rat AF488 were applied (Abcam, Cambridge, MA). The samples were incubated with DAPI. For C3 staining, the tissues were incubated with rabbit anti-mouse C3 overnight at 4 °C. Slides were washed and goat anti-rabbit IgG AF555 was applied. Mounting was performed using Prolong Antifade something Diamond. Images were captured using EVOS FL Cell Imaging System (Thermo-Fisher Scientific) and confocal microscopy.

Tissue for immunohistochemistry was obtained at day 5 of wound healing experiments. A biopsy punch was used to obtain wound areas, which were frozen and cut in OCT compound (Tissue Tek, Sakura).
Cryosections were fixed with 4 % PFA. Unspecific binding sites were blocked with Avidin/Biotin‐Blocking‐Kit (Vector Laboratories) and with protein blocking reagent (ROTI®ImmunoBlock, Roth) supplemented with 5 % BSA (PAN‐Biotech) and 20 % goat serum (Vector Laboratories). The slides were subsequently permeabilized with 0.1 % triton X and were incubated with primary antibodies against F4/80 (BM8, BioLegend), Cd68 (Polyclonal, abcam), iNos (Polyclonal, abcam), Cd163 (TNKUPJ, Thermofisher), Cd206 (polyclonal, abcam) and Arginase‐1 (polyclonal, Novus Biologicals). Goat ant‐rabbit AF488 (Invitrogen), goat anti‐rat biotin followed by streptavidin‐Cy3 (both BioLegend), goat anti rat AF488 (abcam) and goat anti‐rabbit Cy3 (Millipore) were used for detection. Nuclei were stained with Hoechst (Life Technologies). Analyses were performed with fluorescence microscopy (Leica DM6000B) and with confocal microscopy (Leica SP8).