Fifteen gastric cancer patients who underwent gastrectomy with combined LN dissection between March 2014 and August 2017 were enrolled in the study. This study was approved by an institutional review board (UC15SISI0180 and KC18SESI0028). The primary tumor specimens were snap-frozen. The frozen section were stained with hematoxylin and eosin (H&E) for histologic examination by board-certified pathologists for the tumor purity (> 70%). After the positive LNs were confirmed by pathologists, the areas with metastatic cancer tissues were identified in the slide that had been stained with H&E. Ten micrometer thick sections were utilized for microdissection. Tissue sections were micro-dissected using a PixCell II LCM system (Arcturus, Mountain View, CA, USA) by the operator. For matched normal genome DNA, patient's blood was also collected in EDTA-treated tumors. The clinicopathologic features of 15 gastric cancers are summarized in Table 1. One case (GC15) is selected to show the H&E section images for primary and one lymph nodes (Supplementary Fig. 1).
Pixcell 2 lcm system
Manufactured by Arcturus
Sourced in United States
The PixCell II LCM system is a laser capture microdissection (LCM) instrument designed for precise and efficient isolation of specific cells or tissue regions from heterogeneous samples. The system utilizes a high-precision laser to selectively identify and capture targeted cells, enabling downstream molecular analysis.
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Lab products found in correlation
Tissue tek oct medium, by Sakura Finetek (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Picopure kit, by Arcturus (1 mentions)
Picopure rna isolation kit, by Qiagen (1 mentions)
Dnase treatment, by Qiagen (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Fitc conjugated goat anti mouse igg, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
7 protocols using pixcell 2 lcm system
1
Gastric Cancer Genomic Profiling Protocol
2
Laser Capture Microdissection of Pancreatic Cells
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We collected frozen sections on membrane-coated slides (PEN®; Leica Microsystems, Deerfield, IL, USA) and stained them with cresyl violet from Applied Biosystems (Foster City, CA, USA) [71 (link)]. We then microdissected samples of epithelial cells from acini; samples of epithelial cells from intralobular ducts, interlobular ducts, and intralobar ducts; and samples of immune cells from clusters around interlobular ducts using the PixCell II LCM System® (Arcturus Bioscience, Mountain View, CA, USA) and Cap-Sure HS® laser capture microdissection caps from Molecular Devices, Sunnyvale, CA, USA. We collected five or six replicate samples of the epithelial segments and five samples of immune cell clusters from each gland; each sample contained approximately 100 cells.
3
Laser Microdissection of Esophageal Tissue
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The human esophagus was embedded in TissueTek OCT medium (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands) and snap‐frozen in liquid nitrogen. The cryostat sections (8 μm) were laser‐microdissected with a PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA).
4
DNA extraction and RNA isolation from breast cancer
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Genomic DNA was extracted according to the procedure of the Biobank of the institution [21 (link)]. Sample quality and quantity were assessed using NanoDrop (Thermo Scientific, Waltham, MA, USA), and molecular weight was checked by electrophoresis in agarose gels. RNA was obtained from epithelial cells from invasive ductal carcinomas samples captured by laser microdissection using the PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA). Only RNA samples with optical density of approximately 2.0 and RNA integrity number >5.0 were used for microarray experiments [22 ].
5
Microdissection and RNA Extraction
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Leaf tissues from Col-0 and atmyb60-1 plants were prepared according to Kerk et al.47 (link) and microdissected using the Pix‐Cell II LCM system (Arcturus Engineering). RNA from LCM‐harvested cells was prepared using the PicoPure kit (Arcturus Engineering), and reverse‐transcribed using the Superscript™ II reverse transcriptase (Invitrogen).
6
Microdissection of Colorectal Cancer Tissues
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For microdissection, colon cancer tissues and paired noncancerous colon tissues were obtained from patients undergoing surgical resection. The sample from the tumor tissue was mounted in Tissue-Tek OCT compound (Sakura Finechemicals, Tokyo, Japan) and frozen. Each sample was then cut into 10–20 serial sections with a thickness of 10 μm, and sections were mounted on uncoated glass slides. The parts containing areas of cancer cells were identified using hematoxylin–eosin staining and then microdissected according to the standard laser capture procedure32 (link), 33 (link) using a PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA/Olympus, Tokyo, Japan). Total RNA was extracted from Laser-captured cell nests by using PicoPure RNA Isolation Kit according to the manufacturer's protocol, including on-column DNase treatment (Qiagen, Chadstone Centre, VIC, Australia).
7
NANOG Immunostaining and Laser Capture Microdissection
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Tissues were cryo-sectioned at 5 µm thickness on a CM3050S cryostat (Leica Microsystems Inc.), followed by standard immunostaining with anti-NANOG antibody (1:100, mouse mAb, cat# MABD24, clone 7F7.1; Millipore). The predicted NANOGP8 protein is very similar, but not identical, to the embryonic stem cell-specific NANOG1 protein. Hence, most anti-NANOG1 antibodies tested react well with the NANOGP8 protein in PCa cells. Consequently, the NANOG1/NANOGP8 proteins are often simply termed NANOG. Sections were further incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:100; Millipore). Rinsed sections were counter-stained with 10 µg/mL 4', 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Normal IgG was used as a negative control. NANOG+ cells (green) were visualized by the PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA). After micro-dissection with a spot size of 7.5 µm and a pulse duration of 1.5 ms (power 50 mW), the dissected region as indicated was collected in a 0.5-ml micro-centrifuge tube and used for western blotting analysis.
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