GSM8067957
Serially prepared sections (previously hybridized with Whole Transcriptome Assay (WTA) probes) were also immunostained with CD3, Ki-67, ɑ-Smooth Muscle Actin and SYTO83 to help with region of interest (ROI) selection. Briefly, Morphology Marker Solution was prepared in the following proportions per slide: 22ul SYTO (Thermo Fisher Scientific, Cat#S11364), 5.5 ul ɑ-Smooth Muscle Actin (NanoString Technologies), 5.5 ul CD3 (NanoString Technologies), 5.5 ul Ki-67 (NanoString Technologies), and 181.5 ul Buffer W for a total volume of 220 ul/slide. Slides were incubated in this solution at room temperature for 1 hour followed by successive 2x SSC washes, after which, the slides were loaded into the NanoString GeoMx Digital Spatial Profiler (DSP) where overview scans were captured at 20x to guide the selection of ROIs.  Slides were prepared for hybridization following a modified RNA FFPE BOND RX Slide Preparation Protocol in the GeoMx NGS Slide Preparation User Manual (NanoString Technologies, MAN-10115).  Briefly, deparaffinization, antigen retrieval, and Proteinase K digestion was performed on a Leica BOND-RX instrument with Bond Dewax Solution, Bond Epitope Retrieval Solution 2, and a 37°C Proteinase K digest at 1mg/ml. Human Whole Transcriptome Assay (WTA) probes (NanoString Technologies, Cat#121401102) hybridized at 4nM each in Buffer R, and incubated in a hybridization chamber for 16 hrs at 37°