Pannoramic viewer software

Manufactured by 3DHISTECH

Sourced in Hungary, United States

Pannoramic Viewer is a software application developed by 3DHISTECH for viewing and analyzing digital pathology slides. The core function of the software is to provide a user-friendly interface for visualizing and navigating high-resolution whole slide images.

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Pannoramic Viewer (134 citations) Pannoramic Viewer Software package (2 citations)

104 protocols using pannoramic viewer software

Quantifying Vascular Morphology in Muscle

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Adductor muscles were embedded in paraffin, and 5 μm thick sections were cut for histological analysis. Smooth muscle cells were stained with primary antibody mouse anti-mouse α-SMA (Dako, 1:1000). Rabbit anti-mouse HRP (Dako, 1:300) was used as the secondary antibody. Slides were scanned with the Pannoramic MIDI digital slide scanner (3DHistech). The number and lumen diameter of α-SMA positive vessels were analyzed by Pannoramic viewer software (3DHistech, version: 2.3) with 20× magnification (3 sections per limb per mouse). The smallest diameter of vessel was measured in the picture as described previously [37 (link)].
Six μm-thick frozen soleus sections (3 sections per limb per mouse) were fixed in ice-cold acetone and stained using primary antibody anti-CD31 biotin (Biolegend, 102503, 1:100) and an avidin–biotin complex (ABC) kit (Vector, Burlingame, CA, USA). Slides were scanned with the Pannoramic MIDI digital slide scanner (3DHistech). Random snapshots (3 per section) were taken by the Pannoramic viewer software (3DHistech) with 40× magnification (6–9 images per limb per mouse). The CD31 positive area was quantified by ImageJ as described previously [38 (link)].

Goossens E.A., Zhang L., de Vries M.R., Jukema J.W., Quax P.H, & Nossent A.Y. (2021). Cold-Inducible RNA-Binding Protein but Not Its Antisense lncRNA Is a Direct Negative Regulator of Angiogenesis In Vitro and In Vivo via Regulation of the 14q32 angiomiRs—microRNA-329-3p and microRNA-495-3p. International Journal of Molecular Sciences, 22(23), 12678.

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Digital Pathology Analysis Pipeline

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IHC stained slides were digitized with the whole-slide Pannoramic MIDI scanner (3DHISTECH Ltd., Budapest, Hungary) at 20× magnification. Digital image analysis was performed on whole sample areas, excluding only detected artefacts and folded and/or broken regions, which were considered uninformative. Ki67, SYP, KANK1, and DOCK8 were analyzed automatically using Pannoramic Viewer (PV) software (3DHISTECH), and their number of positive cells were determined by applying the NuclearQuant module. This module could be applied to non-nuclear stained samples by adjusting nucleus size settings to detect entire cells when required (nuclear radius 3–15 µm) and adjusting color deconvolution settings. HistoQuant module of PV was applied in VN-stained sections to obtain the areas of each VN intensity expression. All data obtained from PV modules were validated with pathologist’s morphological assessment of the digital image. Digitally obtained data and subsequent pathologist evaluation differed by only 5–10%.

Monferrer E., Sanegre S., Martín-Vañó S., García-Lizarribar A., Burgos-Panadero R., López-Carrasco A., Navarro S., Samitier J, & Noguera R. (2020). Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models. International Journal of Molecular Sciences, 21(22), 8676.

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Quantitative Analysis of Hydrogel Composition

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HE, Ki67 and PTBP1 stained sections were digitalized with the whole-slide Pannoramic MIDI scanner (3DHISTECH Ltd., Budapest, Hungary) at 20x magnification. Detected artefacts and folded and/or broken regions were considered uninformative and were excluded. HistoQuant module was applied in HE-stained sections to obtain the solid area of hydrogels, defining solid area as the regions of the sample with cellular clusters and/or biopolymers. The number of cellular clusters, their respective areas and total hydrogel area for each condition were obtained by segmentation with Pannoramic Viewer (PV) software (3DHISTECH) (Supplementary Fig. 2). Cell nucleus size and shape were also obtained with the HistoQuant module of the PV software. Ki67 and PTBP1 were also determined using PV software, and their expression-related parameters (number of positive cells) were analyzed automatically applying the NuclearQuant module.

Monferrer E., Martín-Vañó S., Carretero A., García-Lizarribar A., Burgos-Panadero R., Navarro S., Samitier J, & Noguera R. (2020). A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior. Scientific Reports, 10, 6370.

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Multiplex Imaging of Tissue Samples

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The slides of the paraffin-embedded tissue were scanned using a Pannoramic Midi Virtual Microscope (3D Histech, Hungary) equipped with a Turrets Chroma 49000/2/4-ET reflector and T-400/495/565 beam-splitter. The cell nuclei were stained with DAPI, DNA barcodes with fluorescein, and nanoparticles with rhodamine. The images were analysed using Pannoramic Viewer software (3D Histech).

Yaari Z., da Silva D., Zinger A., Goldman E., Kajal A., Tshuva R., Barak E., Dahan N., Hershkovitz D., Goldfeder M., Roitman J.S, & Schroeder A. (2016). Theranostic barcoded nanoparticles for personalized cancer medicine. Nature Communications, 7, 13325.

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Quantifying Conjunctival Goblet Cells

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At the end of the study, eyeballs were exenterated then harvested and fixed in formalin for 24 h. Eyes were sectioned 5 μm thick and stained with Periodic Acid Schiff (PAS) to identify goblet cells in the conjunctiva. The histology section images were scanned using a Zeiss Axio Scan Z1 (Thornwood, NY). Numbers of goblet cells were quantified using Pannoramic Viewer software (3D HISTECH Ltd.) of Zeiss Axio Scanned slides at 20×.

Ratay M.L., Glowacki A.J., Balmert S.C., Acharya A.P., Polat J., Andrews L.P., Fedorchak M.V., Schuman J.S., Vignali D.A, & Little S.R. (2017). Treg-recruiting microspheres prevent inflammation in a murine model of dry eye disease. Journal of controlled release : official journal of the Controlled Release Society, 258, 208-217.

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Immunohistochemistry of Tumor and Liver Samples

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Immediately upon removal, tumors and livers were placed in 4% paraformaldehyde (PFA) solution. Then 48 h later, the samples were transferred to 70% ethanol, and they were further processed, embedded, sectioned, and stained with H&E at the University of Ottawa Histology Core Facility. Formalin-fixed, paraffin-embedded slides were deparaffinized in xylene and rehydrated through a 100%–70% ethanol gradient. Heat-induced epitope retrieval was performed at 110°C for 12 min with citrate buffer (pH 6.0), and 3% H₂0₂ was used to block endogenous peroxidases. Sections were further blocked with Background Sniper blocking reagent (BioCare Medical, CA, USA) and incubated with rabbit anti-HA antibody (Cell Signaling Technology, C29F4; 1:1,000 liver, 1:2,000 tumor) and/or rabbit anti-HAdV-5 (Abcam, ab6982; 1:2,000) for 1 h at room temperature. Antibody binding was visualized using the MAHC4TM + DAB detection system according to the manufacturer’s instructions (BioCare Medical). Slides were then counterstained with hematoxylin, and images were obtained using the Zeiss Mirax Midi whole-slide digital scanner. Images were analyzed using Pannoramic Viewer software (3DHISTECH, build 1.15.4.43061, Budapest, Hungary), and they were compiled using Inkscape software.⁷⁴

Del Papa J., Petryk J., Bell J.C, & Parks R.J. (2019). An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo. Molecular Therapy Oncolytics, 14, 107-120.

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Bright-field Histological Image Digitization

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Stained slides were digitally scanned using a bright-field Pannoramic MIDI scanner with a 20X objective from 3DHistech (Budapest, Hungary) and image preparation was done using the 3DHistech Pannoramic Viewer software.

De Angelis P.M., Schjølberg A.R., Hughes J.B., Huitfeldt H.S., Norheim Andersen S, & Østvold A.C. (2018). Nondysplastic Ulcerative Colitis Has High Levels of the Homologous Recombination Repair Protein NUCKS1 and Low Levels of the DNA Damage Marker Gamma-H2AX. Inflammatory bowel diseases, 24(3).

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Quantitative Microscopy Techniques for Reproductive Analysis

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PAS-stained and TUNEL slides were digitized using Pannoramic Flash 250 (3DHistech) with 20×/0.8 NA air objective. Images were produced and analyzed using the Pannoramic Viewer software (3DHistech). Immunofluorescence images of testis sections were produced using an SP8 confocal microscope (Leica Microsystems) with 63×/1.4 NA oil-immersion objective. Chromosome spreads were imaged on an AX70 epifluorescent microscope (Olympus) equipped with a CoolSNAP MYO CCD camera (Photometrics) using a 100×/1.35 NA oil-immersion objective.
All histological and cytological data are representative of ≥2 biological replicates (independent mice) of similar ages. Presence/absence of developing follicles in mutant ovaries (Fig. 3d, e) was assessed by examining ≥30 sections (spanning ≥150 μm of ovarian tissue). Quantitative data were acquired by manual scoring. For TUNEL quantification (Fig. 3c), ≥3 testis sections (≥50 μm apart) were scored per mouse. For detection of number of foci (Fig. 5, 6), immunofluorescence staining was adjusted to be above background level (region containing no cells) using Fiji (Schindelin et al. 2012 (link)) and only foci that overlapped with SYCP3 staining (axis-associated foci) were counted. Spermatocytes were staged on the basis of SYCP3 staining patterns. Statistical analyses were done using GraphPad Prism 7.

Petrillo C., Barroca V., Ribeiro J., Lailler N., Livera G., Keeney S., Martini E, & Jain D. (2020). shani mutation in mouse affects splicing of Spata22 and leads to impaired meiotic recombination. Chromosoma, 129(2), 161-179.

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Immunohistochemical Analysis of Adductor Muscle

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5-μm-thick paraffin-embedded cross-sections of adductor muscle were re-hydrated, and antigen retrieval was performed using citrate buffer. SMCs were stained using primary antibodies against α-SMA (DAKO), ECs were stained with CD31 antibodies (BD PharMingen), and leukocytes were stained using CD45 antibodies (Abcam). Alexa Fluor 647, Alexa Fluor 488, and Alexa Fluor 594 antibodies (Life Technologies and Invitrogen [Alexa Fluor 594]) were used to visualize SMCs, ECs, and leukocytes, respectively. Finally, sections were mounted in Fluoroshield with DAPI (Sigma-Aldrich). The Pannoramic MIDI digital slide scanner (3DHistech) was used to create high-resolution images of the adductor muscles. Snapshots were taken using the Pannoramic viewer software (3DHistech) with 20× magnification. The amount and size of α-SMA positive collaterals was measured using ImageJ (ImageJ 1.48v, NHI).

Welten S.M., de Vries M.R., Peters E.A., Agrawal S., Quax P.H, & Nossent A.Y. (2017). Inhibition of Mef2a Enhances Neovascularization via Post-transcriptional Regulation of 14q32 MicroRNAs miR-329 and miR-494. Molecular Therapy. Nucleic Acids, 7, 61-70.

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Tumor Proliferation Analysis in Hypoxic Microenvironment

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Animals received 200 mg/kg bromodeoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO, B5002) 1 hour prior to termination. Immunolabeled (PanCK, BrdU, DAPI) and scanned (Pannoramic Scanner, 3D-Histech Ltd., Budapest, Hungary) frozen sections of SPC111 tumor colonies were divided into four quarters according to the supply of the nutrients (Q1: area of the tumor facing the lung, Q2: area of the tumor facing the diaphragm, Q3, Q4: area of the tumor facing the vascular proliferations located at the two sides of the sectioned tumor colonies. Proliferating (BrdU labeled) and all tumor cells (DAPI) were counted (Pannoramic Viewer software, 3D-Histech Ltd., Budapest, Hungary). The proliferation index (PI) was defined according to the next formula: PI (%) = (number of proliferating cells/number of all cells) ×100. Presence of vascular proliferations and lack of intratumoral vessels was monitored by appropriate serial sections (CD31, laminin) from at least 4 different depths of the tumor nodules. Samples containing intratumoral vessels were omitted. The average size of the examined tumors was 1,034±255 × 355±77 µm. Statistical significance of difference between the PIs of the four quarters was analyzed by using Student’s t-test. Results were considered as significant at P≤0.05.

Kovacs I., Bugyik E., Dezso K., Tarnoki-Zach J., Mehes E., Gulyas M., Czirok A., Lang E., Grusch M., Schelch K., Hegedus B., Horvath I., Barany N., Megyesfalvi Z., Tisza A., Lohinai Z., Hoda M.A., Hoetzenecker K., Pezzella F., Paku S., Laszlo V, & Dome B. (2022). Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow. Translational Lung Cancer Research, 11(6), 991-1008.

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