Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tumours. Deparaffinised tissue sections underwent antigen retrieval using citrate buffer (pH 6) in a pressure cooker. After endogenous peroxidase blocking, the specimens were incubated with the investigated primary antibodies (anti-LC3B—1:100, #3868, CST; anti-cleaved caspase-3—1:1000, #99664, CST and anti-TOM20—1:500, #42406, CST) at room temperature. Next, Novolink Polymer Detection System (Novocastra) and DAB staining (Aligent, Santa Clara, CA, USA) were used. Slides were counterstained with haematoxylin. Immunostainings were evaluated and documented by two independent observers using Panoramic Viewer Software (3D Histech). Fluorescent stainings were performed using MitoTracker CMXRos (1:10000, #M7512, Invitrogen) and LC3 (1:400, #3868, CST) or TOM20 (1:500, #42406, CST) immunolabelling (after 4% paraformaldehyde fixation, incubation with primary and subsequently secondary goat anti-rabbit Alexa488 IgG antibodies (1:400, #A27034, Thermo Fisher Scientific) and Hoechst (1:600, #H3570, Invitrogen) stainings were used). The immunostainings were analysed using confocal or fluorescent microscopy (Zeiss LSM780. Jena, Germany—Zen Software, Jena, Germany or Nikon Eclipse E600, Nikon Corporation, Tokyo, Japan—Lucia Cytogenetics Software, Laboratory Imaging, Prague, Czech Republic).
Panoramic viewer software
Manufactured by 3DHISTECH
Sourced in Hungary, Germany, Japan
The Panoramic Viewer software from 3DHISTECH is a digital pathology application designed for the visualization and analysis of whole slide images. The software enables users to view, navigate, and examine digital pathology samples in a panoramic format.
Automatically generated - may contain errors
Lab products found in correlation
Panoramic digital slide scanner, by 3DHISTECH (3 mentions)
Novolink polymer detection system, by Leica (2 mentions)
Panoramic midi scanner, by 3DHISTECH (2 mentions)
Hvf22cl 3ccd, by Hitachi (2 mentions)
Bx61vs, by Olympus (2 mentions)
Mirax microscope, by Zeiss (2 mentions)
Panoramic scan, by 3DHISTECH (2 mentions)
Lsm 780, by Zeiss (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Mitotracker cmxros, by Thermo Fisher Scientific (1 mentions)
Calci clear rapid, by National Diagnostics (1 mentions)
Panoramic 250 flash system, by 3DHISTECH (1 mentions)
Cy3 conjugated goat anti rabbit igg secondary antibody, by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Sl038, by Solarbio (1 mentions)
Bl604a, by Biosharp (1 mentions)
Ab230950, by Abcam (1 mentions)
Collagen 1, by Abcam (1 mentions)
α smooth muscle actin, by Abcam (1 mentions)
44 protocols using panoramic viewer software
1
Immunohistochemical Analysis of Cellular Markers
2
Immunohistochemical Analysis of Oral Cancer
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Oral cancer patient tissue samples were obtained and analyzed, abiding the rules and approval of Mackay Memorial Hospital’s institutional review board and the IRB of the Institute of Biomedical Research, Academia Sinica. In brief, the slides with tissue sections were deparaffinized in xylene for 7 min, twice, and then rehydrated in graded ethanol serially from 100% to 70%, followed by rinsing with distilled water. The deparaffinization process was conducted by the Pathology Core of IBMS, Academia Sinica. Subsequently, the slides were immersed in citrate buffer (0.01 M, pH 6.0) and boiled for 50 min in a cooker for antigen retrieval, followed by cooling for 30 min at ambient temperature. Then, the slides were rinsed in distilled water again before immunohistochemistry was performed. The staining procedure was conducted using the Novolink Polymer Detection System (Leica Biosystems, Wetzlar, Germany) following the manufacturer’s instructions. The primary antibody dilution for both the IFIT2 and CD24 was used at a ratio of 1:100. Anti-CD24 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Then, the immunostained tissue section slides were scanned using a Panoramic 250 Flash II whole slide scanner at high magnification. The expression levels of IFIT2 and CD24 were analyzed using the 3DHISTECH Panoramic viewer software (3DHISTECH Ltd., Budapest, Hungary).
3
Histological Evaluation of Dentine Bridge Formation
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Specimens were immersed in a 4% paraformaldehyde solution for 24 hours at 4℃. Samples for demineralization were placed in a decalcifying agent (Calci-Clear Rapid, National Diagnostics, Atlanta, GA, USA) at 4℃ for a month. The tissues were treated with ethanol dehydration, embedded in paraffin, and cut into 5 µm thick sections. Tissue samples were stained with hematoxylin and eosin for evaluation of dentine bridge formation and pulpal inflammation. Slides were scanned with a Panoramic MIDI scanner (3DHISTECH, Budapest, Hungary), and digital image analysis was performed with Panoramic Viewer software (3DHISTECH).
4
Immunohistochemical Identification of CD4+ T Cells
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To identify CD4+ T cells, colon sections were obtained from Foxp3eGFP reporter mice and reacted with rabbit anti-CD4 mAb (Servicebio, Wuhan, China) at 1:400 dilution at 4°C overnight and then incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody for CD4 detection. All slides were counterstained for nuclei with DAPI (1:5000 dilution, Sigma, St. Louis, MO, United States). Slides were observed using the Panoramic 250 Flash system (3DHISTECH, Budapest, Hungary) with Panoramic Viewer software (3DHISTECH, Budapest, Hungary).
5
Immunohistochemical Analysis of VISTA Expression
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IHC was performed on the tissue microarray slides. The tissue microarray slice was cut into 4um and baked in an oven at 60° C for 60 minutes. After dewaxing, hydrogen peroxide blocking, citric acid (BL604A, Biosharp) antigen retrieval, and goat serum (SL038, Solarbio) blocking for 30 minutes at 37° C, the slides were incubated overnight with anti-human VISTA (1:500, ab230950, Abcam) at 4° C. They were then washed by Phosphate Buffered Saline-Tween three times and incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody for 2 hours at room temperature. Thereafter, the slides were stained with 3, 30-diaminobenzidine (DAB) and counterstained with hematoxylin. The images were scanned with a Panoramic Digital Slide Scanner (3DHISTECH) and analyzed using Panoramic Viewer software (3DHISTECH).
6
Determination of Cardiac Ventricular Morphology
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For determining the wet weight of the heart, the right ventricle was carefully dissected from the LV and septum, and both parts of the heart were weighed separately. The relative weight of the right ventricle was expressed as [weight (right ventricle)/weight (left ventricle + septum)].
Histological analysis was performed using formalin-fixed, 5 μm thick paraffin-embedded heart and lung tissues. Cardiomyocyte hypertrophy was measured using cardiac sections that were immunostained using an anti-dystrophin antibody (Merck, Darmstadt, Germany). Mean cardiomyocyte cross-sectional area was separately determined for the right and left ventricles in digitized sections using the Panoramic Viewer software (3DHISTECH Ltd., Budapest, Hungary); Around 400 cardiomyocytes were analyzed per heart from at least four mice per group.
Histological analysis was performed using formalin-fixed, 5 μm thick paraffin-embedded heart and lung tissues. Cardiomyocyte hypertrophy was measured using cardiac sections that were immunostained using an anti-dystrophin antibody (Merck, Darmstadt, Germany). Mean cardiomyocyte cross-sectional area was separately determined for the right and left ventricles in digitized sections using the Panoramic Viewer software (3DHISTECH Ltd., Budapest, Hungary); Around 400 cardiomyocytes were analyzed per heart from at least four mice per group.
7
Histological Analysis of Cardiovascular Tissues
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Following perfusion with saline, hearts, kidneys and aortas were fixed in buffered formalin, embedded in paraffin, and sectioned according to standard procedures. All tissues were stained with hematoxylin and eosin, α-smooth muscle actin, Sirius red, F4/80, CD45, collagen 1 and collagen 3, (all Abcam, Cambridge, UK). Images were captured at 20x magnification, using a Panoramic Scanning 250 microscope and analysed blindly using Panoramic Viewer software (3D Histech Ltd., Budapest, Hungary). Five images of tissues were captured per mouse and staining was quantified as percentage of total area according to published methods using Image J software [17 (link)].
8
Quantitative RNA-ISH Detection of CASC2a
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Tumor and normal tissues were all detect using QuantiGene® ViewRNA ISH tissue assay (Panomics Srl, Vignate-Milano, Italy) for RNA-ISH in line with the manufacturer’s instructions. After deparaffinized, sections were boiled for 5 min in pretreatment solution and digested for 10 min with proteinase K. Next, the sections were hybridized with a custom-designed QuantiGene ViewRNA probe against human CASC2a for 3 h at 40°C. A no-probe sample was used as a control. Slides were stored overnight in storage buffer after the probe hybridization. The next day, hybridized probes were amplified from Affymetrix using PreAmp and Amp molecules. Alkaline phosphatase-conjugated oligonucleotide probes were added and Fast Red was used as substrate to produce signal (red dots) (Pierce, Rockford, IL, Staffs, USA). Slides were counterstained with Hematoxylin and scanned using a Zeiss Mirax Midi Slide Scanner with fluorescence scanner (Centre for Microscopy and Image analysis, UZH, Irchel). Tissue sections were analyzed with Panoramic Viewer software (v. 1.15.2, 3DHISTECH Ltd, Budapest, Hungary,).
9
Immunohistochemical Analysis of Kidney Tissues
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Tissue sections mounted on microslides were deparaffinized in xylene, hydrated through ethanol series, and then dipped in 3% H2O2 to eliminate endogenous peroxidase activity. For antigen retrieval, we microwaved all slides for 15 minutes in Tris-EDTA buffer (pH 9.0) prepared in distilled water. Next, the slides were incubated for 2 hours at 37℃ with antibodies against α-smooth muscle actin (α-SMA) (eBioscience, Tokyo, Japan) and ED-1, a marker of macrophages (1：500, AbD Serotec, Oxford, UK). After irrigation, the sections were incubated at ambient temperature for 30 minutes using a biotin-free polymeric horseradish peroxidase–linker antibody conjugate system (Dako, Glostrup, Denmark). After the slides were washed, chromogen development was performed for 10 minutes. The slides were counterstained with Meyer’s hematoxylin and mounted using Immu-mount (Fisher Scientific, Geel, Belgium). For imaging analysis, we photographed four randomly selected kidney fields per rat in both the cortex and the medulla (40 kidney fields per group). Images were recorded at 40×magnification using the Panoramic Viewer software (3DHISTECH, Budapest, Hungary). All images were evaluated using Adobe Photoshop CS2 for quantification of signals. Outcomes of proliferation are presented as ED-1–positive area and α-SMA area as a percentage of the total area in each field (the cortex and the medulla).
10
Immunostaining Protocol for Spinal Cord
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The slices were treated with 0.3% Triton X‐100 for 20 min and were blocked with serum for 1 h. Then the slices were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: TOM20 (1:400, 11802‐1‐AP, Proteintech, China), anti‐MAP2 (1:400, 8707T, Cell Signaling Technology, USA), anti‐NeuN (1:400, ab104224, Abcam, UK), anti‐beta III Tubulin (1:200, ab78078, Abcam, UK), polyclonal anti‐Cx36 (1:200, 51‐6200, Thermo Fisher, USA), MAP2 Monoclonal Antibody (1:200, 67015‐1‐Ig, Proteintech, China), Anti‐HO‐1 (1:400, ab189491, Abcam, UK). The sections were incubated with a secondary antibody (1:400) at room temperature for 1 h and finally stained with DAPI. Images were obtained using a fluorescence microscope (BX53, Olympus Corporation, Japan) and a confocal microscope (Nikon ECLIPSE Ti‐S, Japan). Panoramic Viewer software (3DHISTECH, Hungary) was used to obtain images of the full spinal cord. The MAP2 fluorescence intensity at high magnification was calculated by Image J.
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