Bond max ihc autostainer

Both anti-CD3 immunolabeling and Masson’s trichrome staining were performed and analyzed by a blinded observer. Kidneys were fixed in 10% neutral buffered formalin, routinely processed and paraffin embedded, then cut to 5 μm sections. Briefly, renal T cell infiltration was analyzed in kidney sections obtained from SGK1^fl/fl controls and SGK1^CD11c KO mice after L-NAME/high salt feeding. All steps besides dehydration, clearing and coverslipping were performed on the Leica Bond-Max IHC autostainer. Slides were deparaffinized. Heat-induced antigen retrieval was performed using the Epitope Retrieval 2 solution for 10 minutes. Slides were incubated with a monoclonal rabbit anti-mouse CD3 antibody at a 1:250 dilution for 60 minutes. The Bond Polymer Refine Detection system was used for visualization. Slides were then dehydrated, cleared and coverslipped. Quantification of CD3 positive cells in the renal cortex was conducted by manually counting the number of immunolabeled cells in 10 consecutive high-power (400X) fields. Mason’s trichrome staining was performed on the Gemini autostainer. Perivascular fibrosis index was assessed at the level of the arcuate arteries and their arteriolar branches throughout the renal cortices on a semiquantitative scale of 0-3.