Thirty-seven tissue samples from murine pancreatic tumors were fixed in 10% neutral buffered formalin and received for histopathology. Tissues were processed routinely, embedded in paraffin, sectioned at 4 mm, and stained with H&E. Five additional unstained sections from each tumor were submitted for immunohistochemical staining with rabbit monoclonal antibodies for pan-macrophage marker IBA-1 (Abcam, clone EPR16589, 1:8,000), and T cell markers: CD3 (Abcam clone SP7, 1:500), CD8 (Cell Signaling Technologies, clone D4W27, 1:400), CD4 (Cell Signaling Technologies, clone D7DZZ, 1:400), and FOXP3 (Cell Signaling Technologies, clone D608R, 1:200) using a Leica Bond Rx autostainer. Whole slide imaging was performed using Leica Biosystems Aperio AT2 digital slide scanner. Digital slides were viewed and representative images were captured at 20 × magnification using Aperio ImageScope Software v12.4.3.7001. Each biomarker was quantified via digital image analysis using tuned algorithms in Leica Image Analysis Software in eSlide manager Spectrum Version 12.4.3.5008. Quantitative data was exported into an excel file and analyzed in GraphPad Prism Software Version 9 via one-way ANOVA; p value < 0.05 was considered significant.
Aperio at2 digital slide scanner
Manufactured by Leica
Sourced in United States
The Aperio AT2 Digital Slide Scanner is a high-performance device designed for digitizing glass microscope slides. It captures high-resolution digital images of the slide contents, enabling the efficient storage, management, and analysis of samples in a digital format.
Automatically generated - may contain errors
Lab products found in correlation
Epitope retrieval solution 2, by Leica (2 mentions)
Mrq 40 antibody, by Cell Marque (2 mentions)
Aperio genie histology pattern recognition software, by Leica (2 mentions)
Bond rx autostainer, by Leica (1 mentions)
Image analysis software, by Leica (1 mentions)
Prism software, by GraphPad (1 mentions)
Ab184032, by Abcam (1 mentions)
Rnascope 2.5 hd assay brown, by Advanced Cell Diagnostics (1 mentions)
Ter119, by BioLegend (1 mentions)
Eslide image management system, by Leica (1 mentions)
Cd45r b220, by BD (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Aperio imagescope software, by Leica (1 mentions)
7 protocols using aperio at2 digital slide scanner
1
Quantitative Analysis of Murine Pancreatic Tumor Immune Infiltrates
2
Evaluating Glomerular Morphology and ACOX1 Expression
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Ten-micron thick sections of formalin-fixed paraffin-embedded kidney were used for haematoxylin and eosin staining, while five-micron thick sections were used for immunohistochemical staining. Scanned haematoxylin and eosin-stained sections were used to measure glomerular area in QuPath (36 (link)), from which glomerular volume was calculated using the Weibel and Gomez formula (37 (link)). Thirty glomerular tufts per sample were manually annotated at random throughout the renal cortex, from a minimum of six animals per experimental group. Kidney sections were stained with anti-ACOX1 antibody (ab184032, Abcam) using the VECTASTAIN Elite ABC-HRP peroxidase kit (PK-6200, Vector Laboratories). An IgG isotype control (ADI-950-231-0025, Enzo Life Sciences) and no antibody control were used to confirm the specificity of staining. All slides were digitized using an Aperio AT2 Digital Slide Scanner (Leica Biosystems).
3
Quantifying Grey Matter Composition
4
In Situ Hybridization of HCV Genotypes
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In situ hybridization assays on liver biopsies were performed using the RNAscope® 2·5 HD-Brown assay (Advanced Cell Diagnostics, Inc., Newark, CA, USA). The detailed information of each probe was: RNAscope® Probe- V-HCV-GT1 (Cat#423221, target region: 36–2399), RNAscope® Probe- V-HCV-GT2 (Cat#423231, target region: 19–2439), RNAscope® Probe- V-HCV-GT3 (Cat#423241, target region: 178–2454), RNAscope® Probe- V-HCV-GT4 (Cat#423251, target region: 103–2529), RNAscope® Probe- V-HCV-GT5 (Cat#423251, target region: 2–2896), RNAscope® Probe- V-HCV-GT6 (Cat#423271, target region: 2–2623), RNAscope® Positive Control Probe-Hs-PPIB (Cat#313901, target region: 139–989 of NM_000942·4), and RNAscope® Negative Control Probe-DapB (Cat#310043, target region: 414–862 of EF191515). The images were acquired using an Aperio AT2 digital slide scanner equipped with a 40× objective (Leica Biosystems Inc., Buffalo Grove, IL, USA). The performance procedure was provided in supplementary information.
5
Quantifying Grey Matter Composition
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Because most of the proteins measured in this study are known to be differentially distributed between grey and white matter, we needed to control for variability in the grey/white matter composition between samples. To accomplish that, 5 μm sections immediately anterior to sections utilized for protein extraction were treated with Epitope Retrieval Solution 2 (Leica Biosystems, Buffalo Grove, IL) for 20 min, immunostained for synaptophysin with rabbit monoclonal MRQ-40 antibody (1:100, Cell Marque, Rocklin, CA), and counterstained with hematoxylin. The sections were then scanned at 40× magnification with an Aperio AT2 Digital Slide Scanner (Leica Biosystems, Buffalo Grove, IL) and subjected to histomorphologic analysis using Aperio GENIE Histology Pattern Recognition software (Leica Biosystems, Buffalo Grove, IL) to differentiate between synaptophysin immunoreactive regions and calculate the grey matter area of each section. We used this procedure to control for potential differences of grey matter area in this analysis cohort.
6
Immunohistochemical Analysis of Mouse Tissues
7
Quantification of Peritumoral Reactive Ductules
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Liver sections were photographed with an Aperio AT2 Digital Slide Scanner (Leica Biosystems Inc., Buffalo Grove, IL). Total EpCAM+ areas within nontumoral tissues were measured using Image J software.30 For quantification of peritumoral RDs, total number of EpCAM+ ductules in 10 randomly selected peritumoral areas within 300 μm of tumor edges were counted and divided by total surface area of captured fields (mm2). RDs were identified based on the following definition by Roskams and Colleagues: reactive ductules with a biliary/HPC phenotype arranged in an irregularly shaped structure.9 For measurement of % paracrine factor+ RDs, % Ki67+ RDs, and % CD34+ area in peritumoral areas, at least four random pictures centered on the EpCAM‐positive RDs were captured. Serial sections of the same area were stained with antibodies for paracrine factors, CD34, and Ki67. The extent of paracrine factor and Ki67 expression by RDs in a given area (0.2025 mm2) was calculated as the ratio of the number of stained ductules to the total number of ductules expressed as a percentage. The area occupied by CD34 and Ki67‐stained cells in intratumoral areas was calculated using Image J software.
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