GSM8067960: Patient_3_20ICH00432TA3_DSP.1012550023361.B.A04; Homo sapiens; OTHER
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°C. Two washes in 50% Formamide/2x SSC (ThermoFisher, Cat#AM9342 / Sigma-Aldrich, Cat#S6639) at 37°C washed non-specifically-bound probes from the slides, which were then blocked prior to antibody staining with 200 ul Buffer W (Nanostring). Regions of interest within the tissue were illuminated with UV light and oligo barcodes were physically aspirated from the tissue and collected into microtiter plates by the GeoMx® Digital Spatial Profiler (DSP) platform. For more information about DSP protocols please see Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x) Each collection of oligo tags from one well (representing an ROI from the tissue section) was indexed with i7xi5 unique dual indexes using GeoMx SeqCode primers with 18 cycles of PCR.  After PCR, indexed AOIs were pooled and purified in two rounds of AMPure XP PCR purification using 1.2x bead:sample ratio.