Capsure macro lcm cap

Cancer tissues from the right ovary and omental sites were collected from a woman with stage IIIc, grade 2/3 serous adenocarcinoma at Northside Hospital (Atlanta, GA) after informed consent was obtained under appropriate Georgia Institute of Technology Institutional Review Board protocols (H14337) according to previously described methods performed in accordance with the relevant guidelines and regulations^{6 (link)}. Briefly, following resection, the tumor tissues were placed in cryotubes and immediately (<1 minute) frozen in liquid nitrogen. Samples were transported on dry ice to Georgia Institute of Technology (Atlanta, GA), and stored at −80 °C. After examination and verification by a pathologist, tissues were embedded in cryomatrix (Shandon, ThermoFisher, Waltham, MA). For each tissue sample, 8 μm frozen sections were cut and attached to uncharged microscope slides. Following dehydration and staining (HistoGene, LCM Frozen Section Staining Kit, Arcturus, ThermoFisher), slides were processed in an Autopix (Arcturus) instrument for laser capture microdissection (LCM). CapSure Macro-LCM Caps (Arcturus) were used to ensure purity of all collected cells. Approximately 30,000 cells were collected for each of the tissue samples.

Six to nine 8.0 μm sections were mounted on uncoated glass slides and stored at −80°C until processed further. For each specimen, one slide was stained with Hematoxylin (Sigma Aldrich, St. Louis, MO) and Eosin (Sigma Aldrich, St. Louis, MO) and examined by a certified pathologist (LL) to confirm the presence of malignant cells. LCM was used to isolate tumor epithelial cells from the surrounding microenvironment as previously described [49 (link)]. Briefly, slides were fixed in 70% ethanol, rinsed with deionized water, stained with Hematoxylin (Sigma Aldrich, St. Louis, MO) and Scott's Tap Water (Electron Microscopy Sciences, Hatfield, PA), and dehydrated in ethanol (70%, 95% and 100%) and xylene. Complete mini protease inhibitors (Roche Applied Science, Indianapolis, IN) were added to the 70% ethanol, deionized water, Hematoxylin, and Scott's Tap Water to preserve protein and phosphorylations from degradation.
Using the infrared laser of a Veritas microdissection system (Arcturus Bioscience, Mountain View, CA) 0.5-18 mm² of malignant cells were collected from each sample on CapSure Macro LCM caps (Arcturus Bioscience, Mountain View, CA).

The LCM process was performed using a PixCell system and CapSure Macro LCM caps (Arcturus Engineering, Mountain View, CA). Single cells or 10 cell pools of cells with positive staining (GFAP+) were lifted individually on caps. HSCs from the first 7 layers of hepatocytes around the portal or central vein were lifted and their position was annotated for future analyses. The annulus for the Laser was adjusted to the size of HSCs (approximately 10 μm, HSCs were lifted and screened for quality as a whole cell on the cap after capture and only accepted if the cell target was fully lifted. During single cell sampling both the tissue and the corresponding cap were inspected for the removed cell body to ensure that the fluoresced HSC of interest is collected. Lysis buffer was added onto the single cell on the cap (5.5 μl; Life Technologies, Grand Island, NY) and cooled on ice before storage at − 80 °C. Hepatocytes, which stained negative for Gfap (Gfap-) and were discernable by size and morphology, were collected according to the same procedure.

The fixation protocol based on snap freezing gave the best results regarding to maintenance of adequate morphology and RNA integrity and was therefore adopted in the further continuation of the LCM protocol development.
The Pixcell IIe IR laser capture (Arcturus engineering, Mountain View, California, USA) was used to microdissect intestinal tissue from 10 CON larvae at 16 dph, ten GF larvae at 10 dph, 10 GF larvae at 16 dph and ten GFPr larvae at 10 dph. Laser settings were chosen to maximize the size of the laser spot without contaminating the sample with non-target tissue. A laser spot size of 30 μm diameter was selected, with 20 mW in power and 5 ms pulse duration. Efficiency of capturing was evaluated by examining excised cell fragments on the CapSure® Macro LCM Caps (Arcturus engineering, Mountain View, California, USA) and the tissue on the slide before and after lifting off the cap. Debris or excess section material was removed to the maximum possible extent by smoothly touching the film with a post-it strip. After tissue collection, the cap was put on a Lo-Bind Microcentrifuge Tube and incubated in 50 μl of RNA extraction buffer. Tubes were kept with the cap down, until all tissue was collected. The whole procedure, from staining to finishing LCM, did not exceed 30 min.

For laser-capture microdissection studies and subsequent RT real-time PCR, frozen sections (8 μ) were obtained from the mammary fat pads, fixed in 70% ethanol, stained with hematoxylin, and progressively dehydrated in 70%, 95%, and 100% ethanol, followed by xylene and air drying. Other frozen sections were stained with hematoxylin directly without fixation. Selected areas which included areas of normal ducts, ducts with hyperplasia, ducts with DCIS, and areas of invasive carcinomas and pulmonary metastasis were microdissected using a Pixcell II Laser Capture Microdissection 788 Laboratory System (Arcturus, Inc., Mountain View, CA) and stored in microdissection caps (CapSure Macro LCM Caps, Arcturus, Inc.) with RNAlater (Thermo Fischer Scientific, Inc.) at − 80 °C.

Laser capture microdissection (LCM) was performed on cryostat cut spinal cord sections (15 µm), attached to RNase-free polyethylene naphthalate (PEN) membrane covered glass slides to facilitate laser cutting as described^{52 (link)}. LCM was carried out with a PALM Zeiss Microbeam microscope using PALM LCM protocols to capture at least 1000 motor neurons per spinal cord onto CapSure Macro LCM caps (Arcturus Bioscience). RNA was extracted using the RNesy micro kit (Qiagen) according to the manufacturer’s instructions.