Archived tissue blocks were obtained and 5μm sections were cut and mounted for analysis. Tissue sections were boiled in EDTA buffer for antigen retrieval. Sections were first stained with rabbit anti-STING (Cell Signaling Technologies, Danvers, MA) and primary antibody binding was detected with HRP conjugated secondary antibodies followed by DAB development and counterstaining. Images were acquired using a Leica SCN400 whole slide scanner. For immunofluorescence staining, sections were stained with anti-CD3 (Spring Bio, Pleasanton, CA) and primary antibody binding was visualized with AlexaFluor 568 conjugated secondary antibodies (Molecular Probes, Eugene, OR) and mounted with DAPI (Invitrogen, Carlsbad, CA) to stain nuclear material. Images were acquired using a Zeiss Axio observer Z1 with attached Nuance Multispectral Image camera and software (Perkin Elmer, Wlatham, MA). All images displayed in the manuscript are representative of the entire section and their respective experimental cohort.
Scn400 whole slide scanner
Manufactured by Leica
The SCN400 is a whole slide scanner manufactured by Leica. It is designed to digitize glass microscope slides and produce high-resolution digital images for various applications. The scanner captures the entire surface of the slide and generates a digital file that can be viewed, analyzed, and shared using compatible software.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Nuance multispectral image camera and software, by PerkinElmer (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti sting, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Opal 520, by PerkinElmer (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Vectra imaging software, by PerkinElmer (1 mentions)
Opal620, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by PerkinElmer (1 mentions)
Matlab software package, by MathWorks (1 mentions)
5 protocols using scn400 whole slide scanner
1
Immunohistochemical and Immunofluorescence Analysis
2
Quantifying HER2+ Breast Cancer Biomarkers
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All cases expressed ER, PR and Ki-67. Four cases expressed p53 and only one case was positive for HER2. Only cases that were positive before decalcification treatment were selected for image analysis. To obtain an adequate cohort of HER2 positive cases, slides were retrieved from the pathology archives under an IRB approved protocol. These HER2+ cases were annotated with HER2 scores from the pathology report: 3 cases with HER2 = 0, 4 cases with HER2 = 1+, 4 cases with HER2 = 2+, and 4 cases with HER2 = 3+. A representative slide was selected by a pathologist (SM) for image analysis. 480 digital images were captured at × 20 magnification from 95 slides on the Leica SCN400 whole slide scanner (Additional file 1 : Table S1). All images were saved as 24bit RGB color images in (*.svs) Leica platform-specific format and stored on a Digital Image Hub (DIH) (Leica Biosystems, Buffalo Grove, IL) for quantification. Using a self fabricated image annotation tool, a pathologist outlined up to 5 randomly selected tumor regions with approximately 2000 cells on each image for quantification (Additional file 1 : Table S1).
3
Immunohistology of Murine Tumor Samples
4
Paraffin Embedding and Immunofluorescence Staining
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Tumors were fixed in Z7 zinc based fixative[31 (link)] overnight. Tissue was then processed for paraffin tissue sections. Tissue was dehydrated through graded alcohol to xylene, incubated in molten paraffin using a Tissue-Tek automated tissue processor (Sakura, Torrance, CA), and then embedded in paraffin. 5 μm sections were cut and mounted for analysis. Trichrome stain was performed according to manufacturer’s protocol (Cardinal Health, Dublin, OH). Primary antibody binding was visualized with Alexa Fluor 488 (Thermo Fisher Scientific), Opal 520, or Opal 620 secondary antibodies (Perkin Elmer, Boston, MA) following either ImmPRESS HRP anti-rabbit or anti-rat polymer incubation (Vector laboratories). Sections were stained with DAPI (Perkin Elmer) for nuclear staining and mounted with FluoromountG (Thermo Fisher Scientific). Images were acquired using: Vectra imaging software (Perkin Elmer); a Leica SCN400 whole slide scanner or a SCN400F fluorescence whole slide scanner. All images displayed in the manuscript are representative of the entire tumor and their respective experimental cohort. NIH image J software was used to separate into their single marker components, to set threshold to minimize back ground noise, and to quantify the positive pixel area in each image. Minimum of 4 tumors per cohort were utilized for analysis.
5
Neuropathological Profiling of Mouse Brains
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Mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS). Brains were drop fixed for 48 h at 4 C in 4% paraformaldehyde (PFA) in PBS. After being cryoprotected and frozen, up to 40 brains were embedded per block in a solid matrix and sectioned coronally at 30 mm (MultiBrain processing by NeuroScience Associates, Knoxville, TN, USA) before being mounted onto slides. All brain sections were stained for AT8, GFAP, Iba1, CD68 and NeuN at NSA using established protocols.
Histological analysis of hippocampus 2 in x 3 in brain array slides were digitally scanned using a Leica SCN400 whole slide scanner (Buffalo Grove, IL). Quantitative image analysis was performed with the MATLAB software package (MathWorks, Natick, MA). For the 5-month time-point, immunolabeled or histochemical stained areas were quantified on 10 serial sections of entire brain hemi-sections for each marker. For the 11-month time-point, regions of interest were manually drawn to restrict the analysis to the hippocampus and analyses were performed on 4-7 serial sections of hippocampus per marker. Quantitative analyses measured the area of immunolabeling of histochemical staining and normalized this area to the total analysis area. The percent of marker area was averaged across serial sections and regions of interest, and reported as an average percent labeled area.
Histological analysis of hippocampus 2 in x 3 in brain array slides were digitally scanned using a Leica SCN400 whole slide scanner (Buffalo Grove, IL). Quantitative image analysis was performed with the MATLAB software package (MathWorks, Natick, MA). For the 5-month time-point, immunolabeled or histochemical stained areas were quantified on 10 serial sections of entire brain hemi-sections for each marker. For the 11-month time-point, regions of interest were manually drawn to restrict the analysis to the hippocampus and analyses were performed on 4-7 serial sections of hippocampus per marker. Quantitative analyses measured the area of immunolabeling of histochemical staining and normalized this area to the total analysis area. The percent of marker area was averaged across serial sections and regions of interest, and reported as an average percent labeled area.
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