Slides scanned at 20× magnification with Aperio ScanScope XT (Leica) were analyzed using Aperio ImageScope Positive Pixel Count software (PPC) (Leica). Parameters to quantify lipid droplet tissue percentage from H&E-stained slides, FOXA1 immunohistochemistry levels, and percent phospho-T156-FOXA2 (p-FOXA2)-positive nuclei are described in Supplementary Tables S3–5 . Stain levels from images of immunohistochemically stained tissue sections taken at 10× magnification were measured with ImageJ software (National Institutes of Health) using the Immunohistochemistry Image Analysis Toolbox(16 (link)) default H-DAB settings (imagej.nih.gov/ij/plugins/immunohistochemistry-toolbox/index.html ) to detect brown staining, then converting the brown stain image to 32-bit grayscale. Quantification parameters are listed in Supplementary Table S6 .
Aperio scanscope xt
Manufactured by Leica camera
Sourced in Canada, United States
The Aperio ScanScope XT is a digital slide scanner designed for high-resolution, whole-slide imaging. It captures high-quality digital images of microscope slides, enabling efficient storage, management, and analysis of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Histogel, by Thermo Fisher Scientific (2 mentions)
Xylene, by Thermo Fisher Scientific (1 mentions)
Toluene solution, by Thermo Fisher Scientific (1 mentions)
Cat hematoxylin, by Biocare Medical (1 mentions)
Benchmark xt autostainer, by Roche (1 mentions)
Imagescope, by Leica camera (1 mentions)
Rm9103, by Thermo Fisher Scientific (1 mentions)
Phospho stat3 y705, by Cell Signaling Technology (1 mentions)
Phospho histone h2ax s139, by Cell Signaling Technology (1 mentions)
Phospho histone h3 s10, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Aperio imagescope software, by Leica Biosystems (1 mentions)
Autostainer xl, by Leica Biosystems (1 mentions)
Masson s trichrome, by Merck Group (1 mentions)
Tissue studio software, by Definiens (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Anti f4 80, by Abcam (1 mentions)
Calponin 1, by Santa Cruz Biotechnology (1 mentions)
Axio scan z1 scanner, by Zeiss (1 mentions)
Dx41 microscope, by Olympus (1 mentions)
19 protocols using aperio scanscope xt
1
Quantifying Lipid Droplets and Transcription Factors
2
Quantifying B7-H3 Expression in Tumor Tissues
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Tumor tissues were stained with the B7-H3 mAb overnight at 4 °C (clone 376.96, 1:1000 dilution, final concentration 1 μg/mL)43 (link), and then stained with HRP polymer conjugated goat anti-mouse secondary Ab (Dako, code K4000, 1:8 dilution) at 25 °C for 1.5 h. Slides were developed using DAB chromogen (Cell signaling), counterstained with CAT hematoxylin (Biocare medical), dehydrated in ethanol, and cleared in xylene (Fisher chemical). Cover slips were added using a histological mounting medium (Fisher, toluene solution). Stained TMA slides were digitally imaged at ×20 objective using the AperioScanScope XT (Leica). TMA slides were de-arrayed to visualize individual cores and each core was visually inspected. Folded tissues were excluded from the analysis using a negative pen, and all other artifacts were automatically excluded with the Aperio Genie software. The B7-H3 positive score was measured using Aperio membrane v9 (cell quantification) algorithm. Percentage of positive cells obtained with this algorithm at each intensity level (negative, low, medium, high) were used to calculate the H-Score using the formula: H-Score = (% at 1+) × 1+(% at 2+) × 2+(% at 3+) × 3. The Aperio color deconvolution v9 algorithm with the Genie classifier was also applied to calculate the area and intensity of the positive stain and generate a Score (0–300).
3
Immunohistochemical Analysis of Organoids
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Organoids were fixed in 10% buffered formalin for 24–48 h and then embedded in Histogel (Thermo Fisher Scientific) prior to processing for paraffin embedding. Paraffin blocks of tumor tissues and organoids were cut at 4 μm thickness, and slices were mounted on charged slides and air dried overnight at 60°C. Sections were stained with hematoxylin eosin (H&E) and by immunohistochemistry (IHC) with various antibodies using the BenchMark XT autostainer (Ventana Medical Systems). Primary antibodies used for IHC analysis included those specific to NRF2 (Abcam Cat# ab137550; RRID: AB_2687540; 1:100; Waltham, MA, USA), NQO1 (Santa Cruz Biotechnology Cat# sc‐271,116; RRID: AB_10611356; 1:100; Dallas, TX, USA), phospho‐S6 (CST Cat#4858; 1:500), TP63 (Dako Cat# M7317; 1:600), and TTF‐1 (Dako Cat# M3575; RICD: AB_2877699; 1:400). Slides were scanned and imaged using the Aperio Scanscope XT (Leica).
4
Quantifying Neurofibrillary Tangles in Entorhinal Cortex
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Hemiforebrains were immersion fixed for 48 hours in 10% neutral buffered formalin in 4°C, blocked coronally into four 3mm slabs using a brain matrix, and processed and embedded into paraffin. Immunohistochemistry was performed on 5 μm coronal sections using automated immunostainers (Ventana Medical Systems, Discovery XT) with nY-29 (Covance, 1:200) mouse monoclonal antibody and counterstained with hematoxylin. After coverslipping, slides were digitized using a slide scanner (Aperio ScanScopeXT, Leica) and numbers of NFT positive neurons were counted in the entorhinal cortex region using image analysis software (Aperio ImageScope) after manual determination of the region of interest.
5
Immunohistochemical Analysis of Tumor Organoids
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EDO grown in Matrigel were fixed in paraformaldehyde for 2 h and embedded in Histogel (Thermo Fisher Scientific, Waltham, MA). Adjacent fresh tumour tissues and Histogel embedded organoids were then fixed with 10% formalin for 24–48 h followed by fixation in 70% ethanol with containing eosin. Formalin-fixed paraffin embedded tumor tissues and organoids were cut into 4 μm thick slices and allowed to dry overnight at 60 °C. Prepared tissue sections were stained with appropriate antibodies using the BenchMark XT autostainer (Ventana Medical System, Tucson, AZ). Primary antibodies specific to p53 (Leica NCL-p53-D07, clone D07), CK7 (Dako M7018, clone OV-TL12130) and HER2 (Thermo Scientific RM9103, Rabbit monoclonal SP3) were used for IHC analysis. The slides were scanned and imaged using an Aperio Scanscope XT (Leica, Canada).
6
Quantifying Cellular Protein Activation
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Immunohistochemistry was performed as previously described [45 (link)]. The following antibodies were used: phospho-STAT3 Y705, phospho-Histone H2AX S139, phospho-Histone H3 S10, and cleaved Caspase 3 (all from Cell Signaling Technology, Danvers, MA). After image acquisition using an Aperio Scanscope XT (Leica), the staining indexes were calculated dividing the number of positive epithelial cells/nuclei by the total number of epithelial cells/nuclei, using Aperio Imagescope software (Leica Biosystems). The resulted ratio is shown as index.
7
Quantification of Metastatic Tumor Burden
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All animals were euthanized by CO2 asphyxiation either at the end of the study or due to tumor burden associated clinical signs such as greater than 20% body weight loss, respiratory distress, or paralysis. All mice underwent a thorough necropsy. Tissues were fixed in 10% neutral-buffered formalin, routinely processed, and embedded in paraffin blocks. For both metastasis models, paraffin blocks of all lungs and tissues noted to have abnormalities at the time of necropsy were sectioned at 5 μm, stained with hematoxylin and eosin (H&E), and microscopically evaluated. Stained sections were scanned into digital format via an Aperio ScanScope XT (Leica, Vista, CA). To calculate areas occupied by metastases, the digital images were annotated by a board-certified veterinary pathologist using Aperio ImageScope software (version 11.2.0.780).
8
Brain Tissue Fluorescence Quantification
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Brain sections were evaluated for GFP fluorescence after counterstaining with DAPI (4′,6-diamidino-2-phenylindole) alone or DAPI with NeuN (hexaribonucleotide binding protein-3).37 (link), 38 (link), 39 (link) Slides were digitized with an Aperio ScanScope XT (Leica) at 200× in a single z plane, with lower magnifications shown as indicated. Fluorescence is quantified using visual assessment and image analysis by brain region, including the cerebrum, cerebellum, pons, hippocampus, thalamus, striatum, and choroid plexus. No GFP or NeuN signal was observed in negative control brain tissue. Fluorescence signal was quantified using a semiquantitative scoring method (visual) and using image analysis.40 (link) Image analysis is reported as number of cells meeting three separate fluorescence thresholds using a scale of 0–3+ as follows: 0: no staining, 1+: mild staining, 2+: moderate staining, 3+: strong staining. An H-score [1] was calculated using QuPath as follows: H-score = [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)].40 (link),41 (link)
9
Histological Analysis of Mouse Brain Tissues
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Brain tissues from the animal were collected at the end of the 3rd- (pretest group) and 9th-month post treatment. Whole mouse brains were fixed in formalin for 48 h, processed, embedded in paraffin, serially sectioned at 5 μm thickness and were used for IHC.
IHC was carried in Bond the fully automated immunostainer (Leica). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Hematoxylin and Eosin (H&E) stain was done in the Autostainer XL (Leica Biosystems Inc., Vista, CA). H&E stained slides were digitally imaged in the Aperio ScanScope XT (Leica) using 20× objective. The complete list of the stains used to evaluate the histological changes 3-month post-irradiation can be found in the Additional file2 .
IHC was carried in Bond the fully automated immunostainer (Leica). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Hematoxylin and Eosin (H&E) stain was done in the Autostainer XL (Leica Biosystems Inc., Vista, CA). H&E stained slides were digitally imaged in the Aperio ScanScope XT (Leica) using 20× objective. The complete list of the stains used to evaluate the histological changes 3-month post-irradiation can be found in the Additional file
10
Masson's Trichrome Staining of Cryosections
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