GSM8206931
Tissue sections were cut at 5 μm thickness and mounted on charged slides. Slides were baked at 60°C for one hour and stored at 4°C in a vacuum-sealed container containing desiccant for up to two weeks. All subsequent steps were performed under RNase-free conditions with DEPC-treated water. Slides were deparaffinized with three sequential five-minute washes in xylenes, followed by two washes in 100% ethanol for five minutes, one wash in 95% ethanol, and one wash in 1X PBS. Antigen retrieval was performed in target retrieval reagent (EDTA, pH 9.0) diluted to 1X in the BioGenex EZ-Retriever System for 10 minutes at 95°C. Slides were then washed with 1X PBS for five minutes. Slides were then incubated in 1 μg/mL proteinase K 10 minutes at 37°C and washed in 1X PBS for five minutes at room temperature. Slides were fixed for five minutes in 10% neutral buffered formalin followed by two washes in NBF stop buffer, for five minutes each, and one wash in 1X PBS for five minutes. Slides were then incubated with UV-photocleavable Human Whole Transcriptome Atlas hybridization probes diluted in buffer R (GeoMx RNA Slide Prep FFPE-PCLN kit) in a hybridization oven at 37°C for 16-20 hours. Following probe incubation, slides were washed with stringent buffer (1:1, formamide:4X SSC buffer) at 37°C twice for 25 minutes each. Then slides were washed twice in 2X SSC buffer. Slides were blocked in 200 μL buffer W (GeoMx RNA Slide Prep FFPE-PCLN kit) for 30 minutes and incubated at 4ºC overnight with SYTO 13 nucleic acid stain (500 nM) and antibodies for pan-cytokeratin (panCK; 1 µg/mL), smooth muscle actin (ACTA2; 1.25 µg/mL), and CD68 (0.5 µg/mL) diluted in buffer W. Slides were washed four times in 2X SSC buffer for 3 minutes each and placed in the nanoString GeoMx DSP instrument. Duplicate 500 µm2 regions of interest (ROI) were selected for each based on fluorescent imaging on the GeoMx DSP instrument (NanoString Technologies). ROI were drawn in the central portion of the endometrium stratum functionalis, to include glandular and not luminal epithelium, for eutopic uterine tissue and in lesions areas with defined epithelium. ROIs were then segmented in order of macrophages (CD68+), epithelium (panCK+), and stroma (ACTA2-) areas of illumination (AOI) for individual collection. AOI were processed sequentially on the instrument using UV light focused through each AOI that released the photocleavable oligos into wells of a 96 well plate. After AOI collection, indexing primers were hybridized during NanoString library preparation. Quality and quantity of the finished library pools were assessed using a combination of Agilent DNA High Sensitivity chips and the QuantiFluor dsDNA System. Spatial Transcriptomics