Histoquant software

Manufactured by 3DHISTECH

HistoQuant is a digital image analysis software developed by 3DHISTECH. The core function of HistoQuant is to provide quantitative analysis of histological images, enabling users to extract and measure various parameters from digital microscopy samples.

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Lab products found in correlation

Caseviewer 3, by 3DHISTECH (2 mentions) Pannoramic midi whole slide scanner, by 3DHISTECH (2 mentions) Pannoramic viewer 1, by 3DHISTECH (2 mentions) Lsm 880, by Zeiss (2 mentions) Paraffin, by Merck Group (1 mentions) Image pro plus version 6, by Media Cybernetics (1 mentions) M0851, by Agilent Technologies (1 mentions) Caseviewer, by 3DHISTECH (1 mentions) Slide converter, by 3DHISTECH (1 mentions) Pannoramic viewer, by 3DHISTECH (1 mentions) Pannoramic midi scanner, by 3DHISTECH (1 mentions) Phospho histone h3, by Cell Signaling Technology (1 mentions) Ab9610, by Merck Group (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Prb 155p, by Fortrea (1 mentions) Prb 160p, by Fortrea (1 mentions) Prb 159p, by Fortrea (1 mentions) Prb 145p, by Fortrea (1 mentions) Ab5694, by Abcam (1 mentions) Ncl ki67p, by Leica (1 mentions)

12 protocols using histoquant software

Histological Analysis of Pulmonary Silicosis

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The left lung was removed, fixed in Milloning buffer solution (pH 7.4) with 4% paraformaldehyde to preserve pulmonary architecture. Briefly, samples were embedded in paraffin (Sigma-Aldrich), and 4 μm-thick sections were cut and stained with hematoxylin and eosin for quantification of granuloma area, Picrosirius for collagen fibers and Sirius Red (pH 10.2) for neutrophils and eosinophils counted in the parenchyma and in peribronchiolar area, respectively. Slides were scanned with 3DHISTECH–Pannoramic MIDI whole slide scanner (capture with a 20× objective lens) and the resulting images analyzed with CaseViewer 3.3, Pannoramic Viewer 1.15.4, and HistoQuant softwares (3DHISTECH). Silica crystals were analyzed, in 15 independent fields, with a light microscope (Olympus BX50) equipped with polarizing attachment for detecting birefringent particles and Image-Pro Plus Version 4.

Ferreira T.P., Mariano L.L., Ghilosso-Bortolini R., de Arantes A.C., Fernandes A.J., Berni M., Cecchinato V., Uguccioni M., Maj R., Barberis A., Silva P.M, & Martins M.A. (2016). Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis. Frontiers in Immunology, 7, 95.

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Histological Analysis of Lung Tissue

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Left lungs from each experimental group were dissected and placed in Millonig fixative solution (pH = 7.4) with 4% paraformaldehyde for 48 h to preserve tissue architecture. Tissues were dehydrated and clarified in xylene before paraffin embedding. Lung sections of 4 μm were stained with hematoxylin and eosin (H&E), to analyze granulomas, or Picrosirius, to evaluate collagen deposition, in light microscope using 3DHISTECH–Pannoramic MIDI whole slide scanner (capture with a 20× objective lens) and the resulting images analyzed with CaseViewer 3.3, Pannoramic Viewer 1.15.4, and HistoQuant softwares (3DHISTECH). Silica crystals were analyzed, in 15 independent fields, with a light microscope (Olympus BX51) equipped with polarizing attachment for detecting birefringent particles and the quantification were performed using the software Image-Pro Plus Version 6.2 (Media Cybernetics Inc., Rockville, MD, United States).

Carvalho V.F., Ferreira T.P., de Arantes A.C., Noël F., Tesch R., Sant’Anna C.M., Barreiro E.J., Fraga C.A., Rodrigues e Silva P.M, & Martins M.A. (2017). LASSBio-897 Reduces Lung Injury Induced by Silica Particles in Mice: Potential Interaction with the A2A Receptor. Frontiers in Pharmacology, 8, 778.

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Morphometric Analysis of Murine Arteriovenous Fistula

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Because most AVF occlusions occur in the venous outflow tract of the AVF, the first 3 venous cross sections downstream of the anastomosis with 150‐μm interval were used for morphological and immunohistochemical analysis. Morphometric analysis of the murine AVFs was performed using Weigert's elastin staining and αSMA (DAKO M0851) as a VSMC marker. Outward remodeling was studied by measuring the length of the internal elastic lamina (IEL) in Caseviewer (3DHISTECH) in Weigert's Elastin stained samples. Tissue within the IEL was defined as IH and determined using histoquant software (3DHISTECH). Luminal area was calculated by subtracting IH from the area within the IEL. αSMA‐positive tissue within the IH was determined using histoquant software. Thrombi were defined as anuclear αSMA‐negative tissue within the IH and calculated by manual tracing using annotations in Caseviewer.
Nuclei of Mac3⁺ cells (BD Pharmingen, 550 292) were counted manually in 3 random fields of view (80× magnification) from which the mean was calculated. Proliferative VSMCs were detected by counting Ki67 (BD Pharmingen, 550 609) and αSMA‐positive cells within the IEL and normalized to the αSMA‐positive area, which was defined using histoquant.

Laboyrie S.L., de Vries M.R., de Jong A., de Boer H.C., Lalai R.A., Martinez L., Vazquez‐Padron R.I, & Rotmans J.I. (2022). von Willebrand Factor: A Central Regulator of Arteriovenous Fistula Maturation Through Smooth Muscle Cell Proliferation and Outward Remodeling. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 11(16), e024581.

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Aortic Histology and Imaging Analysis

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Whole aortas were excised proximal to the aortic arch and femoral bifurcation. Aortas were fixed with 10% formalin overnight and stored in PBS at 4°C. Aortas were cleaned of extraneous fat and tissue, longitudinally dissected and stained with 60% Oil red O. The stained aorta tissues were kept in fixation solution until images were captured. Images were captured digitally using a ColorView 12 CCD attached to an Olympus BX-51 with a 1.25x objective lens and a wide-angle condenser.
All of the aortic histologic images corresponding to the shear wave elastographic region of interests were transformed into digital microscopic images by a scanning system at high resolutions and converted into MRXS files by slide Converter (3DHISTECH, Budapest, Hungary). Computer graphic analysis was performed with Pannoramic Viewer and HistoQuant software (3DHISTECH) under detailed measurement settings Note that the analyzed area on shear wave elastography was the same size as that on histologic examination. The upper half of the heart with the ascending aorta was dissected and paraffin-embedded. Sequential 5 μm thick sections were cut from the heat toward the aortic root and stained with H&E.

Jhun J., Kwon J.E., Kim S.Y., Jeong J.H., Na H.S., Kim E.K., Lee S.H., Jung K., Min J.K, & Cho M.L. (2017). Rebamipide ameliorates atherosclerosis by controlling lipid metabolism and inflammation. PLoS ONE, 12(2), e0171674.

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Immunohistochemical Staining of FFPE Tissue and Neural Tube Patterning

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Immunohistochemical staining on formalin-fixed, paraffin-embedded (FFPE) tissue sections was performed as described previously (Hoelzl et al., 2015 (link)). Briefly, tissue was harvested and fixed in 4% PFA overnight, followed by embedding and sectioning. The following primary antibodies were applied: Phospho-Histone H3 (Ser10, #9701, Cell Signaling, 1:1000), Ki67 (NCL-Ki67p, Leica Biosystems, 1:3000), Trp63 (4A4, sc-8431, Santa Cruz Biotechnology, 1:500), K5 (PRB-160P, Covance, 1:5000), K14 (PRB-155P, Covance, 1:5000), K10 (PRB-159P, Covance, 1:1000), Loricrin (PRB-145P, Covance, 1:200), and alpha-SMA (ab5694, Abcam, 1:500). Analysis of air space and lung tissue was conducted using the Pannoramic MIDI scanner and HistoQuant software (both 3DHISTECH). For assessing neural tube patterning, E9.0 embryos were fixed in 4% PFA for 3 hours or overnight and subsequently immersed in 30% sucrose solution before embedding in Tissue-Tek® OCT (optimal cutting temperature) solution. Samples were cryo-sectioned at 12 μm thickness and incubated with the following primary antibodies: Nkx2.2 (74.5A5, DSHB, 1:20), Pax6 (HPA030775, Sigma, 1:1000), Olig2 (AB9610, Millipore, 1:1000), FoxA2 (4C7, DSHB, 1:10), and Nkx6.1 (F65A2, DSHB, 1:100). Alexa Fluor secondary antibodies (Invitrogen) were used for detection with the Zeiss LSM880 laser-scanning microscope.

Hoelzl M.A., Heby-Henricson K., Gerling M., Dias J.M., Kuiper R.V., Trünkle C., Bergström Å., Ericson J., Toftgård R, & Teglund S. (2017). Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model. Developmental biology, 429(1), 132-146.

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Immunohistochemical Profiling of Microglia

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PBS-perfused brains were immersion-fixed in 4% PFA for 24 h, then sucrose protected (20%) for at least 24 h and then embedded in OCT cryomount (Histolab) and frozen in isopentane. Sections were stained using the following antibodies: Iba-1 (Wako), F4/80-biotin (Cl:A3-1, AbD Serotec), GFP (Abcam, ab13970), GFAP (Abcam, ab7260), Ki67 (Abcam, ab15580), P2ry12 (generated by Dr. O. Butovsky), ICAM-1 (Abcam, ab119871). P2ry12-staining and CX3CR1⁺/Ki67⁺ cell counting was performed Pannoramic viewer and Histoquant software (3D histech).

Lund H., Pieber M., Parsa R., Han J., Grommisch D., Ewing E., Kular L., Needhamsen M., Espinosa A., Nilsson E., Överby A.K., Butovsky O., Jagodic M., Zhang X.M, & Harris R.A. (2018). Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells. Nature Communications, 9, 4845.

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Differential Expression Analysis of IGROV1 Cell Lines

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To analyze any differential expression in the IGROV1 cell lines, a generalized linear model likelihood ratio test was used. Specifically, a negative binomial generalized linear model was fitted, with shRNA-wise or gene-wise dispersions estimated using the Cox-Reid profile-adjusted likelihood method. Then, a likelihood ratio test was employed to check the differential expression^{50 (link)}. The dispersion estimation, model fitting, and testing were implemented using the functions estimateDisp(), glmFit () and glmLRT(), respectively, in the edgeR package^{51 (link)}To interpret the testing results in a biological context, gene ontology (GO) enrichment analysis and KEGG pathway enrichment analysis were employed as downstream procedures, using the online available tool Enrichr^{52 (link)}.
For pathological analysis, IHC images were analyzed by Histoquant software (3D Histech).
The remaining experiments were repeated at least three times and representative data/images are shown. Statistical analysis was performed using GraphPad Prism software, presented as mean ± SEM. For comparisons between two groups, P values were calculated. P values of 0.05 (*), 0.01 (**),0.001 (***) and 0.0001(****) were considered statistically significant.

Zorea J., Prasad M., Cohen L., Li N., Schefzik R., Ghosh S., Rotblat B., Brors B, & Elkabets M. (2018). IGF1R upregulation confers resistance to isoform-specific inhibitors of PI3K in PIK3CA-driven ovarian cancer. Cell Death & Disease, 9(10), 944.

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Spinal Cord Injury mRNA Therapy Analysis

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Three spinal cords from each group were processed for immune- and histochemistry (eGFP/GFAP, eGFP/TUBB3, eGFP/GSA-B4, hIL-10/GFAP, hIL-10/TUBB3, and hIL-10/GSA-B4). The measurement was performed in a 6,500-μm-wide and 1,500-μm-high rectangle-shaped frame around the injected area. The evaluation was performed by HistoQuant software (QuantCenter multiple-module image analysis platform; 3DHISTECH). The animals that received eGFP-encoding mRNA were investigated at 6 time points (1, 2, 5, 9, 14, and 21 d) after injection, and the hIL-10 mRNA-treated rats were only examined on days 1, 2, and 5 after injection. Data were expressed as percentage of colocalized area of marker / total area of marker ratio.

Gál L., Bellák T., Marton A., Fekécs Z., Weissman D., Török D., Biju R., Vizler C., Kristóf R., Beattie M.B., Lin P.J., Pardi N., Nógrádi A, & Pajer K. (2023). Restoration of Motor Function through Delayed Intraspinal Delivery of Human IL-10-Encoding Nucleoside-Modified mRNA after Spinal Cord Injury. Research, 6, 0056.

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Automated Quantification of Cell Populations

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eGFP⁺ (donor‐derived) and Iba1⁺/eGFP⁻ (microglial) cells were counted using HistoQuant software (3D Histech) in all six sections from each mouse. Automated counting was optimised against a manual count of three single sections, to avoid false positives and negatives, until 95% accuracy was reached. Objects in eGFP⁺ stained untransplanted wild type (WT) controls (n = 8) were counted to obtain a mean background value which was subtracted from the mean of the transplanted groups. To assess proliferation, Ki67⁺ cells were counted in six consecutive sections using HistoQuant software, with staining around the choroid plexus and on the very edge of the sections excluded, and validated against manual counting as above. The manual count showed an overestimation of Ki67⁺ cells for each group, therefore a percentage overestimation was determined and removed from the data generated by the software.

Yu K., Youshani A.S., Wilkinson F.L., O'Leary C., Cook P., Laaniste L., Liao A., Mosses D., Waugh C., Shorrock H., Pathmanaban O., Macdonald A., Kamaly‐Asl I., Roncaroli F, & Bigger B.W. (2018). A nonmyeloablative chimeric mouse model accurately defines microglia and macrophage contribution in glioma. Neuropathology and Applied Neurobiology, 45(2), 119-140.

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Histological Assessment of Liver Fibrosis

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Mouse liver tissues were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin (HE). Collagen deposits were stained with sirius red (saturated picric acid containing 0.1% direct red 80 and 0.1% Fast Green FCF). In addition, the samples were immunohistochemically stained using antibodies against S100A4, GS, cytokeratin 19 (Abcam), Egr‐1 (Santa Cruz Biotechnology), F4/80 (Invitrogen), CBP (Cell Signaling Technology), P300 (GeneTex), and Ki‐67 (DAKO) using the VECTASTAIN Elite ABC Kit or the M.O.M Immunodetection Kit (Vector Lab). For human liver‐tissue staining, antibodies against CBP (Cell Signaling Technology), P300 (GeneTex), and Egr‐1 (Cell Signaling Technology) were used. Diaminobenzidine tetrahydrochloride was used as a peroxidase substrate, and sections were counterstained with hematoxylin.
To quantify sirius red–positive areas, standardized computer‐assisted image analysis was performed.^[²²^] An independent pathologist blindly selected five sirius red–stained parenchyma spots in all biopsy samples and automatically measured the sirius red–positive areas using the HistoQuant software (3DHISTECH).

Kimura M., Nishikawa K., Osawa Y., Imamura J., Yamaji K., Harada K., Yatsuhashi H., Murata K., Miura K., Tanaka A., Kanto T., Kohara M., Kamisawa T, & Kimura K. (2022). Inhibition of CBP/β‐catenin signaling ameliorated fibrosis in cholestatic liver disease. Hepatology Communications, 6(10), 2732-2747.

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