RNA in situ hybridization

RNA in situ hybridization was performed on PFA-fixed paraffin-embedded tissue sections using the RNAscope 2.5 HD detection reagent protocol (Advanced Cell Diagnostics, Newark, CA) with accommodation to simultaneously stain for CD3 and CD4 proteins. The 4-μm sections were baked at 60°C for 30 min. Slides were treated with xylene, followed by 100% ethanol, and allowed to dry. Slides were treated with hydrogen peroxide at RT for 10 min and then washed with deionized (DI) water. Samples were treated with RNAscope 1× Target Retrieval Reagent (Advanced Cell Diagnostics, catalog no. 322000) at 102°C for 15 min and then washed. Slides were incubated with deoxyribonuclease I (bovine, Sigma-Aldrich, catalog no. D5319-500UG), to remove DNA fragments, at 37°C for 30 min in a HybEZ Oven II (Advanced Cell Diagnostics, catalog no. 321720) and then washed. RNAscope Protease Plus (Advanced Cell Diagnostics, catalog no. 322331) treatment was applied at 40°C for 30 min and then washed. After target probe amplification and hybridization steps, sections were stained with Fast RED reagent (RNAscope 2.5 HD Detection Reagents–RED, Advanced Cell Diagnostics, catalog no. 322360). Slides were washed with DI water and then PBST [18 g of NaCl and 3 ml of Triton X-100 (Thermo Fisher Scientific, catalog no. BP151-500) to 1 liter of 0.2 M PBS and 1 liter of distilled water]. Slides were blocked with 5% serum in PBST for 1 hour at RT. Tissue sections were then stained according to the immunofluorescence protocol described above.