Donkey anti rabbit igg dylight 550

Salivary glands were extracted from mice and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Frozen tissue slices of 5-μm thickness were fixed with 4% paraformaldehyde for 10 min and rehydrated in PBS. Slides were blocked (10% normal donkey serum in 0.25% Triton-X in PBS) for 30 min at 23 °C. 1:200 dilutions of rabbit anti-human CD3 (Novus Biologicals, Littleton, CO, USA) and goat anti-human PD1 (R&D Systems, Minneapolis, MN, USA) were prepared in the blocking solution and incubated with the tissue for 12 h at 4 °C. Slides were washed three times with PBS for 15 min. Secondary antibodies were prepared as 1:1000 Donkey-anti Rabbit IgG DyLight 550 and 1:4000 Donkey anti-Goat IgG DyLight 488 (Novus Biologicals, Littleton, CO, USA) and incubated with the tissue for 1 h. Slides were washed two times with PBS for 30 min. Slides were mounted with Vectashield antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Burlingame, CA, USA) and coverslipped before imaging with the Nikon A1RS system.

Tumor, salivary gland, spleen, and lung tissue were embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Frozen tissues were sectioned at 5-μm thickness and fixed with ice-cold acetone for 10 min before being rehydrated in PBS for 5 min. Slides were blocked with 10% normal donkey serum in 0.25% Triton-X in PBS for 1 h at room temperature. For primary antibodies, 1:200 dilutions of rabbit anti-human CD3 (Novus Biologicals, Littleton, CO, USA) and goat anti-human PD-1 (Novus Biologicals) were prepared in the blocking solution and incubated with the tissue for 12 h at 4 °C. Slides were washed three times with PBS for 15 min at room temperature. For secondary antibodies, 1:1000 donkey-anti rabbit IgG DyLight 550 (Novus Biologicals) and 1:2000 donkey anti-goat IgG DyLight 488 (Novus Biologicals) were made using the blocking solution. The slides were incubated with secondary antibodies for 1 h at room temperature. Next, slides were washed three times with PBS for 10 min before mounting using a mixture of Fluoromount (Novus Biologicals) and Vectashield with DAPI (Vector Labs, Burlingame, CA, USA). The Nikon A1RS system was used to image slides.