Consecutive sections of the FFPE block were cut on a microtome at 7-μm thickness and mounted on glass slides with polyethylene naphthalate membranes (Thermo Fisher Scientific LCM0522). Slides were immersed 20 s each in xylenes×3, 100% ethanol×3, 95% ethanol×2, 70% ethanol×2, water, hematoxylin (Dako S3309), water, bluing reagent (Thermo Fisher Scientific 7301), water, 70% ethanol×2, 95% ethanol×2, and 100% ethanol×3, xylenes×3. Slides were dissected immediately after staining. Cells were dissected on an ArcturusXT LCM System using both the ultraviolet (UV) laser to cut out each sample and the infrared laser to adhere it to a CapSure HS LCM Cap (Thermo Fisher Scientific LCM0215). Roughly 500 cells were captured by area, according to density estimates by cell counting on small areas. After LCM, the cap was sealed in a 0.5-mL tube (Thermo Fisher Scientific N8010611) and subjected to Smart-3SEQ or DNA library preparation.
N8010611
Manufactured by Thermo Fisher Scientific
The N8010611 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is designed for scientific analysis and research applications. The core function of this product is to perform measurements and data collection, but further details on its intended use are not provided.
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3 protocols using n8010611
1
Laser Capture Microdissection of FFPE Tissue Samples
2
Laser Capture Microdissection Protocol
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Slides were dissected immediately after staining. Histology was categorized by a board-certified pathologist (R.B.W.) according to initial sections stained with hematoxylin and eosin. Cells were dissected on an ArcturusXT LCM System using both the ultraviolet (UV) laser to cut out each sample and the infrared laser to adhere it to a CapSure HS LCM Cap (Thermo Fisher Scientific LCM0215). For bulk samples, roughly 500 cells were captured by area, according to density estimates by cell counting on small areas. For single cells, a cell was dissected from the same area as the corresponding bulk sample, then any additional cells adhering to the cap were ablated with the UV laser. For the ablation validation experiment, two regions of both types (still roughly 500 cells each) were captured on the same cap, and then one region or the other was ablated with the UV laser, except the no-ablation controls (therefore they had roughly 1000 cells). After LCM, the cap was sealed in a 0.5 mL tube (Thermo Fisher Scientific N8010611) and stored at −80°C until library preparation, which was performed within 3 d of dissection.
3
Laser-Capture Microdissection of FFPE Samples
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