GSM8423605: GRN| 006 | CD68+; Homo sapiens; OTHER
Clinically obtained Formalin Fixed Parafin Embeded (FFPE) lung biopsy samples were obtained and assembled into a tissue microarray consisting of 8 individual FFPE lung biopsy samples that were each ~1cmx 1cm in size. Sample processing was done in conjunction with the Nanostring Technology Access Program (TAP) lab, following the the GeoMx DSP user manuals for GeoMx DSP slide preparation (MAN-10150-05), GeoMx DSP sample collection (SEV-00087-05), GeoMx DSP library preparation (MAN-10153-05) and GeoMx DSP data analysis (SEV-00090-05). In brief, tissue slides were baked at 60°C for 1h in a drying oven after which they were deparaffinized and rehydrated using a Leica Biosystems FFPE BOND RX. RNA targets were exposed using proteinase K solution followed by fixation using 10% NBF. After unloading the tissue slides from the Leica Biosystems BOND RX, they were incubated overnight with the RNA probe mix (Whole Transcriptome Atlas, which consists of in situ hybridization probes with UV photocleavable oligonucleotide barcodes). Tissues were then washed and stained with the visualization markers: panCK-488 at 1:500 (Invitrogen, NBP2- 33200AF488), ABCA3-647 at 1:100 (Sevenhills, WMAB17G524 conjugated with the Alexa-647 labeling kit, Thermo A20186), CD68-594 at 1:400 (Santa Cruz, sc-20060AF647) and SYTO 83 at 1:25 (Fisher, S11364). Tissue slides were then loaded in and scanned with the GeoMx DSP instrument. In conjunction with our pathologist, we selected three alveolar macrophage predominant regions of interest (ROIs) per sample using the immunohistochemistry markers on the slide and the H&E staining of the neighboring slide as guide. Serial UV illumination of each ROI was used to collect the probe barcodes from the different ROIs. Using the i5/i7 dual-indexing system of Illumina, each ROI was uniquely indexed.