The whole stained slides for PRMT1, CARM1, ZEB1, and TWIST1 were scanned with Pannoramic DESK Scanner (3DHISTECH Ltd., Hungary) and viewed with Pannoramic Viewer 1.15.4 (3DHISTECH Ltd). The immunohistochemically stained slides were scored by 2 pathologists (IMG and VT). The rest of the slides were scanned and viewed using the Bliss imaging system with WebSlide Browser 4 (both from Bacus Laboratories, Inc., Lombard, IL, USA). Each core was scored separately by dividing the number of positive epithelial cells by the total number of epithelial cells to define the percentage of positive cells in increments of 10 (ie, 0, 10, and 20 etc). At least 100 cells were evaluated in each core. The intensity of staining was scored as 1+, 2+, and 3+. The percentage of positive cells was then multiplied by the intensity of staining and a final score ranging from 0 to 300 was calculated. The mean expression of all cores per case was then evaluated. Nuclear and cytoplasmic staining were separately evaluated when present.
Pannoramic desk scanner
Manufactured by 3DHISTECH
Sourced in Hungary
The Pannoramic DESK scanner is a high-performance digital slide scanner designed for laboratory use. It is capable of capturing high-resolution digital images of glass microscope slides. The device is designed for efficient and accurate digitization of pathological samples.
Automatically generated - may contain errors
Lab products found in correlation
Caseviewer software, by 3DHISTECH (3 mentions)
Fsx100 microscopy system, by Olympus (3 mentions)
Operetta cls high content analysis system, by PerkinElmer (2 mentions)
Pannoramic viewer 1, by 3DHISTECH (1 mentions)
Dylight 488 conjugated goat anti rat igg antibody, by Abbkine (1 mentions)
Cell death detecting kit, by Roche (1 mentions)
Anti brdu, by BD (1 mentions)
Anti 53bp1, by Novus Biologicals (1 mentions)
Cresyl violet acetate, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
N butyl acetate, by Merck Group (1 mentions)
Biotinylated anti rabbit igg, by Vector Laboratories (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Ethanol, by Merck Group (1 mentions)
Histofluid, by Marienfeld (1 mentions)
Pannoramic viewer software, by 3DHISTECH (1 mentions)
Frozen sectioning machine, by Leica (1 mentions)
Cy3 conjugated donkey antirabbit igg h l, by Wuhan Servicebio Technology (1 mentions)
Fitc conjugated goat anti rabbit igg h l, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
12 protocols using pannoramic desk scanner
1
Immunohistochemical Scoring of Protein Markers
2
Immunofluorescence Staining of Cells
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Cells given the designed treatment were seeded on glass slides in a liquid thin layer cell smear, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 5% normal goat serum for 1 h at room temperature. The slides were incubated with primary antibodies at 4 °C overnight, followed by incubation for 1 h at room temperature with Dylight 488-conjugated goat anti-rat IgG antibody (Abbkine, Beijing, China). The slides were washed with PBS and mounted with DAPI. Images were acquired with Pannoramic DESK Scanner (3DHistech Ltd., Budapest, Hungary) and viewed on CaseViewer version 2.3.
3
Comprehensive Cardiac Tissue Analysis
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Slides were processed using Hematoxylin and eosin (H&E) and Masson’s trichrome staining to assess heart structure and fibrillar collagen infiltration. Wheat germ agglutinin (WGA) staining were used to evaluate cardiomyocytes contour following the manufacturer’s instructions (Servicebio). Images were captured using a Pannoramic DESK scanner (3D HISTECH). Additionally, the fibrotic area ratio and myocardial cross-sectional area were quantified using ImageJ software.
4
Quantifying Neural and Cell Death in Ischemic Cortex
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Immunofluorescent staining with the appropriate primary antibodies (anti-NeuN and anti-DCX) and TUNEL staining with an in-situ Cell Death Detecting kit (Roche Diagnostics GmbH, Penzberg, Germany) were performed as previously described (Peng et al., 2019 (link)). Briefly, the brain sections were incubated with primary antibodies at 4°C overnight. After washing with PBS for three times, the sections were incubated with the appropriate secondary antibodies conjugated to fluorescein isothiocyanate. DAPI (1 μg/ml) was then used to counterstain the nuclei. For TUNEL staining, the brain sections were incubated with the TUNEL reaction mixture in the chamber at room temperature, and after washing with PBS, the slices were stained with DAPI. Optical and metric analysis of the stained tissues was performed with a Pannoramic DESK scanner from 3D-HISTECH (Hungary). All of the stained cells in the image were counted, including NeuN+, Sox2+ and TUNEL+ cells. Stained cells in five lesioned regions of the ischemic cortex were counted randomly.
5
Immunofluorescence and Immunohistochemistry Protocol
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For IF and IHC, primary antibodies were incubated ON at 4 °C, followed by subsequent incubation with the secondary antibody for 1 h at room temperature. After antibody exposure, slides were washed twice with PBS. Stained samples were mounted with Mowiol®40-88. IHC were recorded using Pannoramic DESK scanner and analyzed with Case Viewer software (3DHISTECH). For IF, tissue-samples/cells were counterstained with 5 μg/ml DAPI for 15 min after secondary antibody application. IF stained slides were recorded using a FSX100 microscopy system (Olympus). For antibodies, manufacturer’s manuals and instructions regarding concentration or buffer solutions were followed. TP53BP1 foci were analysed in 5 regions of interest (ROI, 10 cells per field) using ImageJ. For ɣ-H2AX, nuclear intensity was measured using ImageJ or the Operetta CLS High-Content Analysis System (Perkin Elmer). Number of cells or fields analysed were indicated
6
Immunohistochemical Analysis of DNA Damage Markers
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Paraffin-embedded sections were deparaffinized and rehydrated as described above. Heat-induced epitope retrieval was performed, and endogenous peroxidase activity was inactivated by hydrogen peroxide. After the sections were incubated in goat serum, the slides were incubated in primary antibodies anti-BrdU (BD, 347580, 1:50), anti-53BP1 (Novus Biologicals, NB100-304, 1:1000), anti-phospho-Chk1 S317 (Bioworld, BS4040, 1:100), anti-phospho-RPA2 Ser4/Ser8 (Thermo Fisher Scientific, A300-245A, 1:1000), and anti-PPP2R2A (CST, 5689S, 1:100) overnight. The slides were then incubated with biotinylated secondary antibody (Citeab, BA-9200, 1:300) and (Citeab, BA-1000, 1:300), incubated with the ABC kit and diaminobenzidine (DAB), counterstained with hematoxylin [38 (link)]. The sections were scanned by a Pannoramic DESK scanner (3DHISTECH Ltd) and viewed by CaseViewer 2.4 software.
7
Nissl Staining for Ischemic Brain Injury
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Nissl staining was performed as previously reported (Lin et al., 2021 (link)). Twenty four hours after reperfusion, the rats (n = 5) were anesthetized and perfused transcardially with cold saline and 4% paraformaldehyde (PFA, Sigma-Aldrich, United States). The brains were rapidly removed, immersed in the fixative for 48 h and then embedded in paraffin and micro-sectioned into coronal slices of 5 μm thickness. The coronal brain sections were stained using 0.5% cresyl violet acetate (Beyotime, Beijing, China). The severity of neural damage was monitored by counting the number of normal neurons in infarct tissues under a Pannoramic DESK scanner from 3D-HISTECH (Hungary). The cells that contained the Nissl stain in the cytoplasm, loose chromatin and prominent nucleoli were considered to be normal neurons. Stained cells in five lesioned regions of the ischemic cortex were counted randomly.
8
Immunohistochemical Analysis of Adenosine A3 Receptor
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Primary antibody (sc-13938, anti-adenosine A3-R) was purchased from Santa Cruz Biotechnology, CA. Secondary antibody (biotinylated anti-rabbit IgG), avidin-biotin complex (Vectastain ABC kit, PK 4001) and aqueous mounting medium (VectaMount) were obtained from Vector Laboratories, CA. DAB substrate kit (ab 94665) was purchased from Abcam (UK). Phosphate-buffered saline (PBS, pH 7.4, 10x solution) was purchased from Morphisto Evolutionsforschung und Anwendung GmbH (Germany). Hydrogen peroxide (H2O2), haemalum, ethanol and n-butyl acetate were purchased from Merck (Germany). Histofluid was obtained from Paul Marienfeld GmbH & Co. KG (Germany), cover plates (24 × 60 mm) were purchased from Menzel-Gläser HistoCom (Austria). All other chemicals were obtained from Sigma Aldrich (Austria). Optical and metric analysis of the stained tissues was performed with a Pannoramic DESK scanner from 3D-HISTECH (Hungary).
9
Immunohistochemistry and Immunofluorescence Protocols
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Standard procedures were used for immunohistochemistry (IHC) and immunofluorescence (IF). For antibodies used in IHC or IF, manufacturer's manuals and instructions were used regarding concentration or buffer solutions. All primary antibodies were incubated overnight at 4°C, followed by washing thrice with PBS and subsequent incubation with the secondary antibody for 1 h at room temperature. IHC slides were imaged using Pannoramic DESK scanner and analysed using CaseViewer software (3DHISTECH). For samples stained with IF, tissue samples/cells were counterstained with 5 μg/ml DAPI, to highlight nuclei, for 15 min after secondary antibody application. Stained samples were mounted with Mowiol® 40–88 and imaged using a FSX100 microscopy system (Olympus).
10
Immunohistochemistry for Breast Cancer Subtypes
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A three micrometer thick section from each TMA paraffin block was stained with haematoxylin and eosin, and based on microscopic analysis they were selected for immunhistochemistry. Because of the external origin of the FFPE, Vimentin (clone V9) was performed first to select the tissues proper for IHC. On the selected specimens, we performed immunohistochemistry for E-cadherin and P-cadherin by using monoclonal mouse anti-human E-cadherin antibody (clone 36B5) and monoclonal mouse anti-human P-cadherin antibody (clone 56C1, Labvision, Fremont, CA, USA). All IHC steps were performed following a protocol selected on MaxBond Autostainer (Leica, Microsystems, Cambridge, UK).
Microscopic analysis and data interpetation. Immunostained slides were scanned with Pannoramic DESK Scanner (3DHistech, Budapest, Hungary) and stored in the Web Slide Library (Case Center, 3DHistech, Budapest, Hungary). Three pathologists analysed the scanned slides by using Pannoramic Viewer Software (3DHistech, Budapest, Hungary) and had high accuracy of microscopic images. They were also able to take pictures from areas of interest. Membrane (M), cytoplasmic (C) or mixed (MC) patterns were assessed and correlated with molecular subtypes of breast cancers. Statistical analysis included data processing with SPSS software version 20 and correlations were considered significant when p was less than 0.05.
Microscopic analysis and data interpetation. Immunostained slides were scanned with Pannoramic DESK Scanner (3DHistech, Budapest, Hungary) and stored in the Web Slide Library (Case Center, 3DHistech, Budapest, Hungary). Three pathologists analysed the scanned slides by using Pannoramic Viewer Software (3DHistech, Budapest, Hungary) and had high accuracy of microscopic images. They were also able to take pictures from areas of interest. Membrane (M), cytoplasmic (C) or mixed (MC) patterns were assessed and correlated with molecular subtypes of breast cancers. Statistical analysis included data processing with SPSS software version 20 and correlations were considered significant when p was less than 0.05.
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