Donkey anti goat af555

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.

The CD1d tetramer immunofluorescence has been described previously (Lee et al., 2015 (link)). Briefly, fresh thymic lobes were incubated with PE-CD1d-PBS57 tetramer at 4°C overnight. The tissues were then washed with PBS and fixed in 4% paraformaldehyde (PFA) for 1 hr and snap frozen in OCT. Five micrometer sections were made and stained with Goat-anti-R Phycoerythrin (PE) (Abcam) followed by Donkey-anti-Goat AF555 (Abcam). The sections were incubated with Rabbit-anti-β5t (MBL International) and anti-CD45.1 AF647 (Biolegend) at 4°C overnight, followed by Goat-anti-Rabbit BV480 (BD biosciences). The sections were counterstained with DAPI covered with Prolong anti-fade mounting medium (Life Technologies). The images were obtained with a Leica DM6000B Epi-Fluorescent microscope.