Dab substrate reaction

Spleen tissues were fixed in 10% buffered formalin solution and cut into paraffin sections using standard methods. Paraffin slides were dewaxed and rehydrated, followed by antigen retrieval as described previously [19 (link)]. Slides were incubated with mouse anti-rat CD4 and anti-rat CD8 (1:50 dilution, Biolegend), rabbit anti-rat programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1; 1:50 dilution, Abcam, Cambridge, MA), rabbit anti-rat cytotoxic T-lymphocyte-associated protein 4 (CTLA4; 1:50 dilution, FabGennix, Frisco, TX), and human leukocyte antigen-antigen D related (HLA-DR; 1:50 dilution, Proteintech, Chicago, IL) antibodies. Normal mouse and rabbit IgG (Vector Labs, Burlingame, CA) were used as negative controls. Slides were washed with Tris-buffered saline (TBS) after primary antibody incubation. Sections were then treated with biotinylated anti-mouse or anti-rabbit IgG followed by Vectastain ABC reagent and DAB substrate reaction (Vector Labs) to reveal positive staining. Slides were counterstained with hematoxylin. No immunostaining was observed on negative control slides.