We quantified migratory tumor cells after different CM treatment using a Boyden chamber transwell assay. We added CM (700 μl per well) to a 24-well companion plate with a 0.8 μM pore size insert (BD Biosciences, San Jose, CA, USA). Further, we seeded 5 × 104 cells/300 μl medium in the upper part of each insert and incubated for 6 hours at 37°C. Subsequently, we washed transwell inserts with phosphate-buffered saline (PBS) and placed them in methanol for fixation, followed by 10-min crystal violet staining. After washing with distilled water, each membrane was mounted on slides with Pertex (Medite GmbH, Burgdorf, Switzerland). The slides were scanned with NanoZoomer 2.0-HT digital slide scanner C9600 (Hamamatsu Photonics). We quantified the number of migratory cells per membrane using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as previously described (6 (link), 7 (link), 41 (link)).
Nanozoomer 2.0 ht digital slide scanner c9600
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer 2.0-HT digital slide scanner C9600 is a high-throughput slide digitization system developed by Hamamatsu Photonics. It is designed to capture high-resolution digital images of microscope slides for various applications, including research and clinical diagnostics.
Automatically generated - may contain errors
5 protocols using nanozoomer 2.0 ht digital slide scanner c9600
1
Quantifying Migratory Tumor Cells by Boyden Chamber
2
Quantitative Analysis of Intestinal Tissue
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Intestinal tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS), embedded in paraffin, and sectioned to a thickness of 3.5 μm. H&E and IHC staining for β-catenin and Ki67 were performed as previously described [3 (link)]. The sections were scanned using a NanoZoomer 2.0-HT Digital slide scanner C9600 (Hamamatsu) and analyzed with NDP.view 2 software. Ki67 positive proliferating cells were quantified as the percentage diaminobenzidine (DAB) positive area per total hematoxylin stained nuclear area, using ImmunoRatio, a publicly available application for quantitative image analysis [33 (link)]. Activated EGFR levels were assessed as described previously [3 (link)], using an antibody against EGFR phosphorylated on Tyr1068 and Alexa Fluor 488 conjugated secondary anti-rabbit antibody, and measuring the fluorescence intensity along a line across colonic crypts. Apoptotic intestinal epithelial cells were detected using the ApopTag® Peroxidase In Situ Oligo Ligation (ISOL) Apoptosis Detection Kit as recommended by the manufacturer (Chemicon), and quantified using Axiovision Software (Zeiss) and manual counting of stained cells per mm2 tissue from 3 randomly chosen mucosal areas. For quantifications, colon sections from 4-5 controls and 4-5 Pacs2-/- mice were used.
3
Cerebellar Neuropathology Analysis Using IHC
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Neuropathologic analysis and immunohistochemistry (IHC) were performed on small
cerebellar sections from formalin-fixed and paraffin-embedded (FFPE) tissue
blocks. Hematoxylin and eosin and a set of primary antibodies, which are
summarized in eTable 1 (links.lww.com/NXI/A726 ), were used to stain the sections. For double
immunolabeling using primary antibodies derived from different species, the same
antigen retrieval techniques were applied (eTable 1). Immunoreactivity was
subsequently visualized by using alkaline phosphatase–conjugated
secondary antibodies for subsequent development with Fast Blue BB salt as well
as biotinylated secondary antibodies and peroxidase-conjugated streptavidin for
subsequent development with aminoethyl carbazole.22 (link) Slide scanning was performed on a NanoZoomer
2.0-HT digital slide scanner C9600 (Hamamatsu Photonics, Hamamatsu, Japan).
cerebellar sections from formalin-fixed and paraffin-embedded (FFPE) tissue
blocks. Hematoxylin and eosin and a set of primary antibodies, which are
summarized in eTable 1 (
immunolabeling using primary antibodies derived from different species, the same
antigen retrieval techniques were applied (eTable 1). Immunoreactivity was
subsequently visualized by using alkaline phosphatase–conjugated
secondary antibodies for subsequent development with Fast Blue BB salt as well
as biotinylated secondary antibodies and peroxidase-conjugated streptavidin for
subsequent development with aminoethyl carbazole.22 (link) Slide scanning was performed on a NanoZoomer
2.0-HT digital slide scanner C9600 (Hamamatsu Photonics, Hamamatsu, Japan).
4
Immunohistochemical Analysis of Brain Markers
5
Quantification of Chemotactic Migration in Cancer Cells
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Chemotactic migration was quantified using a Boyden chamber Transwell assay. Cancer cells (A549, H226, HCC15, H1650 and A427) or PBMC-derived macrophages were seeded in the upper part of a chamber (8.0 μm pore size; BD Biosciences) at a density of 0.05 × 106 cells/chamber in 300 µl DMEM. CM (700 µl/ well) was distributed to each well of the 24-well companion plates (BD BioSciences). Serum-free and serum-rich media were used as migration controls. Cells were incubated for 8 h at 37 °C. Then the chambers were washed with PBS and placed in methanol for fixation, followed by 10 min staining with crystal violet. After a final wash with H2O, each membrane was mounted on a slide with Pertex (Medite GmbH, Burgdorf, Switzerland), slides were scanned with a Nanozoomer 2.0HT digital slide scanner C9600 (Hamamatsu Photonics). The number of migrated cells per membrane was counted using ImageJ software.
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