GSM9533163: 001Non immune_2; Homo sapiens; OTHER
TMA Slides were processed and profiled on the GeoMx Digital Spatial Profiler (DSP) as per manufacturer's protocol (Nanostring™ GeoMx manuals). Briefly, FFPE slides were baked on a heating pad at 60°C for 30 minutes and deparaffinized sequentially in xylene, 100% ethanol, 95% ethanol and PBS. Antigen retrieval was performed at 99°C in a steamer, followed by incubation with Proteinase K (1µg/mL, Ambion) at 37°C. Samples were washed and fixed in 10% Neutral Buffered Formalin (Thermo Scientific™ Richard-Allan Scientific™ 5700). In situ hybridization with the human WTA probes from the GeoMx assay kit was set up overnight in the hybridization chamber at 37°C as per manufacturer's protocol. Samples were immunostained with a cocktail of morphology markers that included CD45 AF532 (2B11+PD7/26, Novus, 19.25µg/mL), CK8/18 AF594 (K8.8+DC10, Novus, 10µg/mL) Nur77 AF647 (Novus, 34µg/ml) and counter-stained with Syto 13 (50µM) for 1hr at room temperature. Slides were profiled on the Digital Spatial Profiler with an exposure time of 300 msecs for CD45 and CK8/18 and 200 msec for Nur77 and nuclear stain. Region of interests (ROIs) including immune and non-immune compartment were defined and collected based on unbiased threshold method based on CD45 immunostain. Taking into account of the high background signal of Nur77 immunostain, Nur77 positivity was manually defined into either Nur77+ or Nur77- TMA cores. Both CD45+ immune regions and CD45- non-immune regions from Nur77+ and Nur77- TMA cores were used for downstream transcriptome profiling. PCR amplification as per GeoMx Human Whole Transcriptome Atlas protocol using Seq Code pack reagents and index plates.