Digital images of 40 μm-thick sections at 20x magnification were captured using a ScanScope XT slide scanner (Aperio) at a resolution of 0.5 μm/pixel. Image quantitation was conducted manually using the Area Quantification and Microglial Activation modules in HALO analysis software (Indica Labs Inc.) as previously described [38 (link), 39 ]. Analysis thresholds were optimized for each immunohistochemical stain (pSer129-αSyn, GFAP, Iba1 and CD68 in the nigra, or striatal TH) to provide broad detection of pathology in regions with both high and low density pathology, without the inclusion of background staining. Tissue sections spanning the ipsilateral and contralateral substantia nigra or striatum were outlined manually and quantified for the positive staining percentage area occupied by the different immunostains with the positive pixel algorithm, sampled across 2–4 randomly selected images per animal. Positive staining of different immunohistochemically-labeled pathologies was set to a baseline threshold on pixel intensity, such that any pixel at that intensity or greater (i.e., darker) was quantified as a pixel-positive area. The number of positive pixels was normalized per area outline for each section to account for outlined region-to-region area variability. All sections/images were batch analyzed in Halo using the same parameters.
Halo analysis software
Manufactured by Indica Labs
Sourced in United States
HALO analysis software is a digital pathology image analysis platform developed by Indica Labs. It provides tools for quantitative analysis of whole-slide images and tissue-based biomarkers.
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9 protocols using halo analysis software
1
Quantitative Neuroimaging Analysis Protocol
2
Multiplex Immunofluorescence Analysis of EBVaGC
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Six cases with EBVaGC were subjected to multiplex immunofluorescence staining. The staining was performed according to the manufacturer’s protocol with the following markers: CD20 (Roche, 760-2531), CD21 (ZSGB-BIO, ZA-0525), CD4 (ZSGB-BIO, ZM-0418), CD8 (ZSGB-BIO, ZM-0508), and FOXP3 (Abcam, ab20034) using a PANO 7-plex IHC kit. Cell nuclei were stained with DAPI (1:2000 dilution). Multiplexed color slides were imaged with a Polaris slide scanner (AKOYA BIOSCIENCES) and five random areas on each sample were analyzed blindly at 200× magnification using HALO analysis software (Indica Labs).
3
Quantitative Assessment of Hippocampal and Cortical Regions
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Slide-mounted horizontal brain sections were scanned using a ScanScope XT slide scanner (Aperio) at a resolution of 0.5 μm/pixel. Annotation and quantitative assessment of the hippocampal and cortical regions of interest (6 complete horizontal brain sections assessed per animal, left/right hemispheric regions were annotated and assessed separately) were performed using HALO analysis software (Area quantification, Object colocalization, and Microglial activation modules; Indica Labs Inc.). For each quantitative approach, threshold parameters were developed and optimized to detect positive-staining without background staining or tissue-level artifacts. All sections/slides were assessed using the optimized HALO batch analysis function for each marker of interest. All annotations and assessments were conducted agnostic to slide identification or animal genotype.
4
Identifying Crown-like Structures via DenseNet
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eWAT sections (n = 3 per group) were stained by H&E, coverslipped and then imaged on an AxioScan Slide Scanner. Images of whole sections were then analysed using HALO analysis software (v3.0.311.201, Indica Labs, Corrales, NM, USA). Specifically, the DenseNet neural network algorithm of the HALO AI module was trained to selectively identify crown-like structures across random images in both groups. Entire sections were then analysed using the trained algorithm while blinded to the groupings.
5
Evaluation of FRA Expression in TNBC PDx Models
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The FRA expression of TNBC PDx models (Oncodesign) was evaluated by IHC staining using the anti‐FRA mAB (clone 26B3), which had been co‐developed as an in vitro diagnostic antibody with BioCare. Formalin‐fixed paraffin‐embedded (FFPE) slides were incubated with anti‐FRA mAb (clone 26B3) or control murine IgG (isotype match [IgG1/κ] of 26B3), and the bound antibody was visualized using the Ultravision Quanto Mouse on Mouse staining kit (Thermo Scientific). The resulting stained slides were scanned on a Pannoramic Midi slide scanner (3DHISTECH). Staining area, excluding areas of poor quality or necrosis, was identified, and the percentage of FRA‐positive cells (FRA‐positive ratio) in the area was digitally measured using the HALO® analysis software (Indica Labs., ver2.0.1018). FRA expression level was estimated through FRA‐positive cell frequency. OD‐BRE‐0631, of which less than 5% of cells expressed FRA, was considered “low‐FRA–positive,” while IM‐BRE‐563, of which 5%‐25% of cells expressed FRA, was considered “high‐FRA–positive.”
6
Quantitative Analysis of Coronal Brain Sections
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Digital scans of slide-mounted coronal sections at ×20 magnification were generated using a ScanScope XT slide scanner (Aperio) at a resolution of 0.5 µm/pixel. Quantitative analyses were performed using HALO analysis software (Area quantification and Object colocalization modules; Indica Labs Inc.) as previously described66 . Briefly, threshold parameters were developed within the modules to provide broad detection of DAB-positive immunostaining without the inclusion of background staining for each marker of interest. Anatomical regions were annotated across a minimum of 3 to 5 coronal sections using the Allen Mouse Brain Atlas (Allen Institute) as an annotation guide67 (link). All sections/images were batch analyzed in HALO analysis software using the developed threshold parameters to detect DAB staining in relation to annotated area (% area occupied). For specific quantification of NeuN staining in the hippocampus, the pyramidal cell layer of the CA1 and CA2/3 subfields, and granule cell layer of the dentate gyrus were annotated to detect total immunostained area across 3 adjacent coronal sections (every 4th section). NeuN-positive area was then normalized to generate a % NeuN-positive area relative to controls.
7
Quantitative Analysis of Hippocampal and Cortical Regions
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Slide-mounted horizontal brain sections were scanned using a ScanScope XT slide scanner (Aperio) at a resolution of 0.5 μm/pixel. Annotation and quantitative assessment of the hippocampal and cortical regions of interest (6 complete horizontal brain sections assessed per animal, left/right hemispheric regions were annotated and assessed separately) were performed using HALO analysis software (Area quantification, Object colocalization, and Microglial activation modules; Indica Labs Inc.). For each quantitative approach, threshold parameters were developed and optimized to detect positive-staining without background staining or tissue-level artifacts. All sections/slides were assessed using the optimized HALO batch analysis function for each marker of interest. All annotations and assessments were conducted agnostic to slide identification or animal genotype.
8
Quantitative Adipose Tissue Analysis
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Formalin-fixed gonadal adipose tissue was processed, embedded and sectioned, and 3 μm sections were stained with haematoxylin and eosin (H&E). Whole sections were scanned using the Axioscan digital slide scanner (Zeiss, Germany). Images were analysed using HALO analysis software (Indica labs, USA) using DenseNet AI Plugin classifiers (‘crown-like structures’ or CLS analysis) and the Vacuole Quantification tool (adipocyte cell size) adapted to the H&E-stained WAT sections. The estimated adipocyte number was calculated based on methods previously described [28 (link)] by dividing the total volume of gonadal WAT (estimated from the tissue weight and density of rodent visceral WAT) by the mean adipocyte volume derived from the cross-sectional area measured by HALO. The percentage adipocytes surrounded by CLS, a marker of adipocyte death [44 (link)], was calculated by normalising the number of CLS by the number of adipocytes per section.
9
Comprehensive Immune Profiling of Tumor Microenvironment
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