Nbp2 33184af532

Slides were prepared by baking in a drying oven and then processed using the Leica BOND platform (Leica Biosystems) as specified by the NanoString GeoMx DSP Slide Preparation User Manual. Briefly, slides were deparaffinized with xylene then rehydrated through a graded ethanol series. Targets were exposed by incubating the slides in a pressure cooker at 100°C in 1X Tris-EDTA buffer, pH9 followed by proteinase K digestion. Next, the GeoMx NGS-RNA Probe Mix (NanoString Technologies) was applied to each slide, and slides were incubated at 37°C for 16-24 hours. This probe mix contained barcoded oligonucleotide probes against 1,700 gene targets and included internal positive and negative control probes. After washing, the slides were then blocked in Buffer W (NanoString Technologies) and stained with morphology markers for 1 hour. The fluorescently conjugated morphology markers used are as follows: Iba1 at 1:75 dilution (EMD Millipore, MABN92-AF647), CD3 at 1:50 dilution (Abcam, ab196147), GFAP at 1:3000 dilution (Novus Biologicals, NBP2-33184AF532), and SYTO 13 nuclei acid stain at 1:5 dilution (Thermo Scientific S7575).

Slides were baked in a drying oven, deparaffinized, rehydrated in an ethanol gradient, and placed in ditheyl pyrocarbonate (DEPC)-treated water. Antigen retrieval was performed with 1X citrate buffer,pH6 in a pressure cooker on high temperature setting for 15 minutes. The slides were then blocked and incubated with the primary antibody mix and the fluorescently conjugated morphology markers. The morphology markers used are as follows: Iba1-647 at 1:75 dilution (EMD Millipore, MABN92-AF647), CD3-594 at 1:50 dilution (Abcam, ab196147), GFAP-532 at 1:3000 dilution (Novus Biologicals, NBP2-33184AF532), and SYTO 13 at 1:5 dilution (Thermo Scientific S7575).