For immunostaining, we used a SARS-CoV-1 monoclonal antibody, previously shown to cross-react with SARS-CoV-2 in mammalian cell lines.46 (link) Immunostaining was performed using the aforementioned primary antibody diluted 1:2000 in blocking buffer (3%BSA in 1x PBS) and a goat anti-Mouse IgG (H+L) Alexa Fluor 555 conjugate (Abcam, catalog number A32727) secondary antibody diluted 1:2,000 in blocking buffer. We acquired fluorescence images at 20x magnification using Metafer Slide Scanning platform (MetaSystems). Raw images were stitched with VSlide software (MetaSystems).
Metafer slide scanning platform
Manufactured by MetaSystems
Sourced in Germany
The Metafer Slide Scanning Platform is a laboratory equipment designed for automated slide scanning. It provides high-resolution digital imaging of microscope slides. The system captures and digitizes slide content for further analysis and data processing.
Automatically generated - may contain errors
Lab products found in correlation
Vslide software, by MetaSystems (3 mentions)
Mayer s hematoxylin, by Agilent Technologies (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Axio imager, by Zeiss (2 mentions)
Axio imager m1 microscope, by Zeiss (2 mentions)
Vslide, by MetaSystems (1 mentions)
Ipgell, by Genostaff (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Blocking reagent, by PerkinElmer (1 mentions)
Ikaros software, by MetaSystems (1 mentions)
Bluing buffer, by Agilent Technologies (1 mentions)
Glycerol, by Merck Group (1 mentions)
Vysis lsi mdm2 spectrumorange probe, by Abbott (1 mentions)
Vysis cep 12 d12z3 spectrumgreen probe, by Abbott (1 mentions)
Zytolight spec mdm2 cen 12 dual color probe, by ZytoVision (1 mentions)
X rad 320, by Precision X-Ray (1 mentions)
19 protocols using metafer slide scanning platform
1
SARS-CoV-2 Immunostaining Protocol
2
High-Resolution Slide Scanning and Preparation
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Slides were scanned under a high-resolution microscope Metafer Slide Scanning Platform (Metasystems) to obtain tissue tile images and software VSlide (Metasystems) to stitch the high-resolution images together. After imaging, the glycerol and cover glass were carefully removed by holding the slides in an 800 mL water beaker and letting the glycerol diffuse until the cover glass detached and density changes were no longer visible in the water. The slides were then dried at 37°C.
3
FISH Analysis of CIC Rearrangement
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FISH analysis was performed on 4-µm-thick sections. A solid cell pellet was prepared from adherent tumour cells by iPGell (Geno Staff). Break-apart probes were used for CIC (custom-made probe; Chromosome Science Labo) genes as previously described12 (link). FISH images were obtained using the Metafer Slide Scanning Platform (MetaSystems). The presence of split 5′ and 3′ signals or isolated 5′ signals in more than 20% of tumour cells was considered positive for CIC rearrangement.
4
Karyotyping Cells with Aphidicolin Treatment
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Cells were treated for 40 hr with 0.2 aphidicolin μM. In the final 90 minutes of treatment, cells were co-incubated with colcemid (0.1 μg/mL). Cells were harvested and incubated in 5 mL 0.075 M KCL for 10 minutes before being fixed with the addition of 1 mL methanol and acetic acid (3:1). Cells were resuspended in 10 mL of methanol and acetic acid (3:1) overnight at 4 °C. Cells were dropped onto wet slides and dried at 42 °C for at least one hr before staining with 8% (v/v) KaryoMAXTM Giemsa in 1x Gurr buffer for three minutes, washing in 1x Gurr buffer for 3 minutes, washing in water for 3 minutes, and drying. Dry slides were then imaged on the Metasystems Metafer slide scanning platform.
5
Fluorescence In Situ Hybridization Assay
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In total, 100 000 LCLs were attached to microscopy slides using Cytospin funnels (500 rpm for 5 min at RT), fixed with 70% ethanol (15 min at 4C) and stored at -20C until the day of the analysis.
2.5 μl of IgH/c-myc translocation dual fusion probe (LPH-041, Cytocell) was added per slide, and slides were incubated in thermal cycler (2 min at 75C then at 37C overnight). Next day, slides were washed with 0.4xSSC (2 min at 72C) and 2xSSC + 0.05% Tween-20 (1 min at RT). Samples were then counterstained with DAPI, and images were acquired using Metafer Slide Scanning Platform (MetaSystems).
2.5 μl of IgH/c-myc translocation dual fusion probe (LPH-041, Cytocell) was added per slide, and slides were incubated in thermal cycler (2 min at 75C then at 37C overnight). Next day, slides were washed with 0.4xSSC (2 min at 72C) and 2xSSC + 0.05% Tween-20 (1 min at RT). Samples were then counterstained with DAPI, and images were acquired using Metafer Slide Scanning Platform (MetaSystems).
6
Immunofluorescence Staining of Cells
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Following fixation for 15 min in ice-cold freshly prepared 4% paraformaldehyde (Merck, Molsheim, France), cells were incubated in TNB Buffer: 0.5g of blocking reagent (PerkinElmer, Stockholm, Sweden) to 100 mL TBS buffer (Tris/NaCl pH 7.4) for 30 min at room temperature. Cells were then incubated with the 1’ab diluted in 0.3% Triton X-100, 0.1% NaN3 in 1X PBS overnight at +4 °C. Following washing, cells were incubated with 2’ab diluted in TNB buffer, for 2 h at room temperature. The cells were washed and mounted with Prolong Gold antifade with DAPI (Thermo Fisher Scientific, Stockholm, Sweden) to visualize the nuclei. Stained cells were analysed using a Metafer® Slide Scanning Platform (version 3.13.4, Metasystems, Heidelberg, Germany). A list of the antibodies and kits used is presented in Table S2 .
7
Chromosome Fragility Quantification
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Chromosome fragility test was performed as previously described.11 (link) Twenty-five metaphases were scored for chromosome breakages using the Metafer Slide Scanning Platform from Metasystems. Results were graphed as distributions of metaphases presenting 0, 1, and >1 chromatid break. Statistical analysis was performed applying chi-squared test.
8
Chromosome Preparation and Analysis
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Chromosome preparation was performed following protocol described previously [31 (link)]. Metaphases were analysed using Metafer slide scanning platform and IKAROS-software (MetaSystems). Twenty metaphases were analysed for each cell line.
9
Tissue Staining with Hematoxylin and Eosin
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Sectioned slides were incubated at 37°C for 1 min., fixed in 3.7%–3.8% formaldehyde (Sigma-Aldrich) in PBS (Medicago) for 10 min, and then washed in 1x PBS (Medicago).
For staining, sections were incubated in Mayer’s hematoxylin (Dako, Agilent, Santa Clara, CA) for 4 min, bluing buffer (Dako) for 30 s, and Eosin (Sigma-Aldrich) diluted 1:5 in Tris-base (0.45M Tris, 0.5M acetic acid, pH 6.0) for 30 s. The slides were washed in RNase and DNase free water after each of the staining steps.
After air-drying, the slides were mounted with 85% glycerol (Merck Millipore, Burlington, MA) and coverslips (Menzel-Gläser). Bright-field (BF) images were taken at 20x magnification using Metafer Slide Scanning platform (MetaSystems). Raw images were stitched with VSlide software (MetaSystems). The coverslip and glycerol were removed after imaging by immersing slides in RNase and DNase free water.
For staining, sections were incubated in Mayer’s hematoxylin (Dako, Agilent, Santa Clara, CA) for 4 min, bluing buffer (Dako) for 30 s, and Eosin (Sigma-Aldrich) diluted 1:5 in Tris-base (0.45M Tris, 0.5M acetic acid, pH 6.0) for 30 s. The slides were washed in RNase and DNase free water after each of the staining steps.
After air-drying, the slides were mounted with 85% glycerol (Merck Millipore, Burlington, MA) and coverslips (Menzel-Gläser). Bright-field (BF) images were taken at 20x magnification using Metafer Slide Scanning platform (MetaSystems). Raw images were stitched with VSlide software (MetaSystems). The coverslip and glycerol were removed after imaging by immersing slides in RNase and DNase free water.
10
Dual-Color FISH for RET Fusions
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First, we performed using a dual-colour break-apart probe for the RET gene (Supplementary Table 2 ; Chromosome Science Labo, Inc., Sapporo, Japan). Among RET gene break-apart probe-positive cases, we next performed using break-apart probes for both KIF5B and CCDC6.
A total of 50 non-overlapping tumour cells with hybridisation signals examined for each case were captured using the Metafer Slide Scanning Platform (MetaSystems, Altlussheim, Germany). The signal in each cell was categorised into one of the following seven patterns: (1) fused 3′/5′ only; (2) fused 3′/5′ and both isolated 3′ and 5′ (split); (3) both isolated 3′ and 5′ (split) only; (4) fused 3′/5′ and isolated 5′ (5) fused 3′/5′ and isolated 3′ (6) isolated 5′ only; and (7) isolated 3′ only. A split signal was defined by 5′ and 3′ probes observed at a distance of greater than one-fold the signal size. A FISH-positive case was defined as ⩾20% of tumour cells having any split signals or any isolated 3′ (red) signals. The threshold for the RET gene was determined in 27 cases, yielding both FISH and previously reported RNA sequence data (Kohno et al, 2012 (link)) (Supplementary Figure 1 ).
A total of 50 non-overlapping tumour cells with hybridisation signals examined for each case were captured using the Metafer Slide Scanning Platform (MetaSystems, Altlussheim, Germany). The signal in each cell was categorised into one of the following seven patterns: (1) fused 3′/5′ only; (2) fused 3′/5′ and both isolated 3′ and 5′ (split); (3) both isolated 3′ and 5′ (split) only; (4) fused 3′/5′ and isolated 5′ (5) fused 3′/5′ and isolated 3′ (6) isolated 5′ only; and (7) isolated 3′ only. A split signal was defined by 5′ and 3′ probes observed at a distance of greater than one-fold the signal size. A FISH-positive case was defined as ⩾20% of tumour cells having any split signals or any isolated 3′ (red) signals. The threshold for the RET gene was determined in 27 cases, yielding both FISH and previously reported RNA sequence data (Kohno et al, 2012 (link)) (
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