Versa slide scanner

Staining was performed using the RNAscope platform (ACD), according to the manufacturer’s protocol for the RNAscope 2.5 HD Detection Reagent (red, 322360) and technical note for fresh-frozen tissue (320536). Tissue sections were fixed with 4% PFA for 15 min at 4°C, and dehydrated with a series of ethanol washes (50%, 70%, 100%, 100%) for 5 min each. The sections were pretreated with hydrogen peroxide for 10 min, washed once with distilled water, pretreated with Protease IV for 30 min at RT and washed with 1X PBS. Sections were then individually hybridized for 2 h at 40°C, with probes targeting either TREM2 (404111, ACD), DapB (negative control; 310043, ACD) or PPIB (positive control; 313911, ACD). After hybridization, sections were washed twice with 1X PBS for 2 min and subject to 6 amplification steps (30 min at 40°C, 15 min at 40°C, 30 min at 40°C, 15 min at 40°C, 30 min RT, 15 min RT) prior to detection. Signal was detected using Fast Red reagent (322360, ACD) for 10 min at RT, and briefly washing with tap water prior to counterstaining with hematoxylin. Slides were mounted using xylene and EcoMount. Images were scanned using a Leica-Aperio Versa slide scanner.