For immunofluorescence (IF), cells were washed, fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were washed with PBS, permeabilized with PBS + 0.1% TritonX-100 for 5 min and blocked with 5% BSA in PBS for 1h at RT. Cells were then incubated with primary antibody diluted in 5% BSA in PBS for 2h-3h at RT. Primary antibody was detected with Alexa Fluor secondary antibody diluted 1:500 in PBS at RT for 1h in the dark. For IF conducted on paraffin embedded tissue, all stainings with primary antibodies were done after deparaffinization, rehydration through a graded series of alcohol, antigen retrieving by boiling for 10 min in Sodium Citrate buffer (pH 6) and blocking in 2% BSA in PBS-T buffer. Antibodies were diluted in blocking solution and incubation was at 4°C overnight. Secondary fluorescent-tagged antibodies were from molecular probes (Invitrogen). Stained cells and sections were scanned using a Pannoramic Midi II digital slide scanner (3DHISTECH Ltd).
Pannoramic midi 2 digital slide scanner
Manufactured by 3DHISTECH
Sourced in Germany, Hungary
The Pannoramic Midi II is a digital slide scanner manufactured by 3DHISTECH. It is designed to digitize glass microscope slides for use in digital pathology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Molecular probe, by Thermo Fisher Scientific (2 mentions)
Pannoramic viewer, by 3DHISTECH (2 mentions)
Senescence cells histochemical staining kit, by Merck Group (1 mentions)
Slideviewer 2, by 3DHISTECH (1 mentions)
Ki67 antibody, by Abcam (1 mentions)
Cleaved caspase 3 asp175 antibody, by Cell Signaling Technology (1 mentions)
Halo image analysis software, by Indica Labs (1 mentions)
8 protocols using pannoramic midi 2 digital slide scanner
1
Immunofluorescence Staining Protocol
2
Senescence Cells Histochemical Staining Assay
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Fresh mouse lung tissues were immersed in a fixation solution containing 2% formaldehyde and 0.2% glutaraldehyde in phosphate buffered saline (PBS) for 45 min, then transferred to 30% sucrose, and fixed overnight. The tissues were cut into 6 μm thick cryosections, which were then fixed and incubated overnight at 37 °C with the staining mixture supplied in the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, St. Louis, MO, USA) before being counterstained with Nuclear Fast Red. Finally, the slides were scanned and photographed with a Pannoramic MIDI II digital slide scanner (3DHISTECH, Budapest, Hungary).
3
Quantifying chronic hemorrhage burden
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Sixteen Stage 2 lesions from 13 acute Krit1+/−Msh2−/− mice and 12 Stage 2 lesions from 9 Cdh5iCreERT2Krit1ECKO mice were stained using Perls Prussian blue to detect non-heme iron deposition, an index of chronic hemorrhage burden.8 (link), 9 (link) The stained tissue sections were then scanned using a 20X Zeiss objective (Zeiss Microscopy, Göttingen, Germany) mounted on a Pannoramic midi II digital slide scanner (3D Histech Ltd, Budapest, Hungary) at the Integrated Light Microscopy Facility (University of Chicago, Chicago, IL, USA), and exported as magnified TIFF images (5X) from the digital Pannoramic Viewer (3D Histech Ltd).
The computational quantification of iron was performed using Image J (National Institute of Health, Bethesda, MD, USA). For each exported magnified TIFF images, a RGB color-deconvolution algorithm was applied to isolate the red, green, blue components.41 (link), 42 (link) The blue component image was then selected, inversed and an automatic Rényi entropy-based thresholding method applied.43 For each Perls Prussian blue-stained section, the cumulative intensity value was assessed based on the clusters that survived the thresholding. The cumulative intensity value was then normalized to the lesional area.
The computational quantification of iron was performed using Image J (National Institute of Health, Bethesda, MD, USA). For each exported magnified TIFF images, a RGB color-deconvolution algorithm was applied to isolate the red, green, blue components.41 (link), 42 (link) The blue component image was then selected, inversed and an automatic Rényi entropy-based thresholding method applied.43 For each Perls Prussian blue-stained section, the cumulative intensity value was assessed based on the clusters that survived the thresholding. The cumulative intensity value was then normalized to the lesional area.
4
Immunofluorescence Staining Protocol
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For immunofluorescence (IF), cells were washed, fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were washed with PBS, permeabilized with PBS + 0.1% TritonX-100 for 5 min and blocked with 5% BSA in PBS for 1h at RT. Cells were then incubated with primary antibody diluted in 5% BSA in PBS for 2h-3h at RT. Primary antibody was detected with Alexa Fluor secondary antibody diluted 1:500 in PBS at RT for 1h in the dark. For IF conducted on paraffin embedded tissue, all stainings with primary antibodies were done after deparaffinization, rehydration through a graded series of alcohol, antigen retrieving by boiling for 10 min in Sodium Citrate buffer (pH 6) and blocking in 2% BSA in PBS-T buffer. Antibodies were diluted in blocking solution and incubation was at 4°C overnight. Secondary fluorescent-tagged antibodies were from molecular probes (Invitrogen). Stained cells and sections were scanned using a Pannoramic Midi II digital slide scanner (3DHISTECH Ltd).
5
Quantifying chronic hemorrhage burden
6
RNAscope Analysis of Thyroid Gene Expression
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Spatial gene expression was evaluated by RNAscope (Advanced Cell Diagnostics Biotechne, Newark, CA, USA) in situ hybridization in thyroid glands exposed to control and 10 µM PFOS (n = 4). The sections were deparaffinized and stained using the RNAscope® 2.5 HD - RED assay (Advanced Cell Diagnostics Biotechne) according to the manufacturer’s instructions with 15 min RNAscope H202 and Protease Plus (Advanced Cell Diagnostics Biotechne) treatment, RNAscope Wash Buffer Reagents (Advanced Cell Diagnostics Biotechne) and reagents provided with the kit (Wang et al., 2012 (link)). The following RNAscope® target probes were used; Foxe1 (Rn-Foxe1-C1), Pax8 (Rn-Pax8-C1), Cdh16 (Rn-Cdh16-C1). Ppib (Rn-Ppib) with and without xylene was used as positive control. Sections were counterstained with Meyer’s haematoxylin. The slides were scanned using a 40x objective in a Pannoramic Midi II digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Images were qualitatively evaluated with the SlideViewer 2.5 software (3DHISTECH Ltd.).
7
Quantification of Fibrotic Area in Liver Tissue
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Slides from human or murine liver tissue were scanned as indicated after Sirius red or after immunhistochemical staining of p70S6K, α-SMA, and collagen 1α1 using a Pannoramic MIDI II digital slide scanner from 3D-Histech (Sysmex, Norderstedt, Germany). The stained fibrotic area was quantified via QuPath software (https://qupath.github.io/ ) and ImageJ2 software (National Institutes of Health, Bethesda, MD) as described previously.35 (link)
8
Evaluating SQLE Protein Expression in OSA Prognosis
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From YEPCOME Biotechnology, we purchased a TMA (Cat YP‐BonSur2201) (n = 79) to analyse the association of SQLE protein expression with the prognoses of OSA patients. In this TMA, two samples were excluded because no sarcoma cells were found in them. Therefore, only 77 samples were analysed further. IHC staining was performed using standard procedures with SQLE antibody (H‐6) (Santa Cruz Biotechnology, Cat sc‐271651). All slides were scanned with a Pannoramic Midi II digital slide scanner (3DHISTECH Ltd.). The demographic characteristics and clinical data of the patients from the TMA are listed in Table S6 .
All tumour tissues from mice were fixed in 4% paraformaldehyde for subsequent paraffin embedding and cut into 5 μm thick sections. IHC staining was performed using standard procedures with SQLE antibody (Proteintech, Cat 12544‐1‐AP), Ki‐67 antibody (Abcam, Cat ab15580), and cleaved caspase‐3 (Asp175) antibody (Cell Signaling Technology, Cat 9661). The details of the antibodies used are listed in TableS5 .
The staining results were analysed with HALO image analysis software (Indica Labs). The average OD was defined as the integrated OD divided by the area of the region of interest (ROI) and used to represent the strength of SQLE abundance.
All tumour tissues from mice were fixed in 4% paraformaldehyde for subsequent paraffin embedding and cut into 5 μm thick sections. IHC staining was performed using standard procedures with SQLE antibody (Proteintech, Cat 12544‐1‐AP), Ki‐67 antibody (Abcam, Cat ab15580), and cleaved caspase‐3 (Asp175) antibody (Cell Signaling Technology, Cat 9661). The details of the antibodies used are listed in Table
The staining results were analysed with HALO image analysis software (Indica Labs). The average OD was defined as the integrated OD divided by the area of the region of interest (ROI) and used to represent the strength of SQLE abundance.
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