GSM6043757
The FFPE tissue blocks were cut at 5um, and the slides were subjected for in-situ mRNA hybridization. The slides were incubated at 60C for 35m and moved into Leica BOND RX for RNA antigen retrieval, using proteinase K (1ug/ml) for 5 minutes and HIER2 for 20m. RNA probes (NanoString GeoMx Mouse Whole Transcriptome Atlas) were added for hybridization at 37 C for 16h. The stringent washes were performed according to the manufacturer's instructions. The slides were blocked with W buffer for 1h and then incubated with primary antibody cocktail for 1h at room temperature. The cocktail contains CD3 (1:100 dilution, Ab135372) and B220 (1:60 dilution, BD #550286) antibodies. The slides were washed with SSC buffer two times and subjected to incubation with secondary antibodies, AF594 Goat anti Rat at 1:100 dilution and AF647 Goat anti Rabbit at 1:100 dilution, for 45 minutes at RT. The slides were washed with SCC buffer twice and incubated with morphology markers cocktail, which contains Pancytokeratin-AF532 (1:40 dilution) and Syto13 (1:10 dilution), for at RT for 1h.  After washing with ASSC buffer twice, the slides were immediately loaded on to the GeoMX DSP and scanned for selection of regions of interest (ROIs). Upon ROI selection, segmentation was performed by masking for desired markers.  ROIs 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. Each collection of oligo tags from one well representing an ROI was indexed with i7xi5 unique dual indexes using GeoMx SeqCode primers with 18 cycles of PCR. After PCR, indexed ROIs were pooled and purified in two rounds of AMPure XP PCR purification and sequenced on the NextSeq 2000 using a P3 flow cell kit (27 x 27 and index 8 x 8).