Formalin-fixed paraffin-embedded sections of human tumor biopsies or xenografts were deparaffinized and either stained with human CD31-specific antibody (clone JC70, Cell Marque, California, USA) or a human CD13-specific antibody (clone SP10087, Cell Marque, USA) using Bench Mark Ultrastainer (Ventana, Roche, Basel, Switzerland). Imaging was perfomed using Vectra® 3.0 system (Perkin Elmer, Germany) and InForm Analysis Software. For H&E staining, formalin-fixed paraffin-embedded sections of xenografts were stained with hematoxylin (Merck, Darmstadt, Germany) for 3 min and eosin (Roth, Karlsruhe, Germany) for 2 min. The tissue sections were mounted with Vitrocloud (Langenbrink, Bissendorf, Germany). Imaging was perfomed using Vectra® 3.0 system (Perkin Elmer, Germany) and InForm Analysis Software. For EVG staining, Formalin-fixed paraffin-embedded sections of xenografts were stained with resorcinol fuchsin (Waldeck, Germany), picrofuchsin (Waldeck, Germany) and hematoxylin according to manufacturer´s instruction using the Tissue Tek Prisma® (Sakura, Düsseldorf, Germany) autostainer. Imaging was performed using Vectra® 3.0 system (Perkin Elmer, Germany) and InForm Analysis Software.
Inform analysis software
Manufactured by PerkinElmer
Sourced in Germany
InForm analysis software is a data analysis tool developed by PerkinElmer. The software is designed to process and analyze data from various laboratory instruments and experiments. It provides users with the ability to organize, visualize, and interpret their data in a efficient and effective manner.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark ultra stainer, by Roche (1 mentions)
Vectra 3.0 system, by PerkinElmer (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Nel791001kt, by PerkinElmer (1 mentions)
Vectra imaging software, by PerkinElmer (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Ab16669, by Abcam (1 mentions)
Vectra 3, by PerkinElmer (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Vectra3 slide scanner, by PerkinElmer (1 mentions)
Phenochart, by PerkinElmer (1 mentions)
Primary antibody diluent, by Leica (1 mentions)
Bond automated immunostainer, by Leica (1 mentions)
Eclipse ci upright microscope, by Nikon (1 mentions)
Nuance multispectral imaging system, by PerkinElmer (1 mentions)
9 protocols using inform analysis software
1
Immunohistochemical Analysis of Human Tumor Xenografts
2
Multiplex IHC Analysis of FFPE Slides
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Five FFPE slides from specimens, with observed TLS in the H&E-stained sections, were subjected to multispectral immunohistochemical (mIHC) staining29 30 (link) using the Opal color kit (PerkinElmer, Hopkinton, Massachusetts, USA), according to the manufacturer’s instructions, to provide simultaneous detection of adaptive immune system cell types, such as CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD45RO+ memory T cells. Cell nuclei were stained with DAPI. Multiplexed color slides were imaged with a PerkinElmer Vectra automated multispectral microscope at 100× magnification and analyzed using PerkinElmer inForm Analysis Software.
3
Quantifying Tumor Infiltrating Immune Cells
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Vectra staining was performed on tumors to identify tumor infiltrating immune cells as previoulsy described [16 (link)]. Bi-flank 4434 murine melanoma tumours grown in C57BL/6 mice were treated with Virus 16 on days 1, 3 and 5, then collected at day 10 after the first injection, fixed overnight in 10% neutral buffered formalin and then transferred to PBS prior to processing and embedding. Tissue sections were labeled with immunofluorescent stains as follows; CD8 (Cat No: 14–0808-82), CD4 (Cat No:14–9766-82), and foxp3 (Cat No: 14–5773-82), all from eBioscience. Images were then quantified by an automated cell segmentation and phenotyping algorithm, using inForm analysis software (Perkin Elmer). Four thousand four hundred thirty-four cells are a murine melanoma tumor cell line generated in house at The Institute of Cancer Research, London.
4
Quantitative Immunohistochemistry of Tumor Tissues
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In vivo lung tissues of tumor-bearing mice and wild-type (WT) littermates were fixed in formalin, embedded with paraffin, and cut into 4-μm-thick sections from paraffin-embedded samples. The formalin-fixed and parrffin-embedded (FFPE) tissue sections were dewaxed and rehydrated through an ethanol gradient. Antigen retrieval was obtained by a pressure cooker in citrate buffer (pH 6.0) for 3-min heating, and endogenous peroxidase was blocked by 3% H2O2 for 10 min at RT. The sections were then incubated with goat serum for 30 min and antibodies for 1 h at RT, after which the slides were rinsed with phosphate-buffered saline (PBS) three times and incubated with secondary antibodies for 10 min at RT. Fluorescent amplification signal solution (1:200) was then used, and DAPI was applied to label nuclear cells. Whole-tissue slides were scanned at 4× magnification performed with Vectra automatic quantitative pathological imaging analysis system by Phenochart panoramic analysis software and inForm Analysis Software (PerkinElmer).
5
Multiparametric Immunohistochemical Phenotyping
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Immunohistochemistry (IHC) was performed as previously reported.52 (link) After deparaffinization, 4.5 µm thick sections were incubated with rabbit anti-CD3 (SP7, Abcam, ab16669, 1:100), followed by treatment with tyramide-fluorophore reagent (PerkinElmer, NEL791001KT; Life Technologies, T20950) at 1:100 dilution in Amplification plus buffer (PerkinElmer, NEL791001KT) for 10 min at room temperature (RT) and washed in Tris-Buffered Saline, 0.1% Tween®20 Detergent (TBS-T) and H20. Similar procedure was performed for the rat anti-CD8 (4SM15, ThermoFisher, 14-0808-82, 1:2000), rat anti-CD4 (4SM95, ThermoFisher, 14–9766, 1:200) and rat anti-Foxp3 antibodies (FJK-16s, ThermoFisher, 145 773–82, 1:100) using an anti-rat secondary HRP (Vector Labs, MP-7444–15). After washes in TBS-T, 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, D1306, 1 mg/mL stock, 1:500 in Phosphate buffered saline (PBS)) was added to slides for 10 min at RT. Slides were imaged at both 4 x, and 20 x using Vectra imaging software (PerkinElmer), and the number of cells were enumerated from the top nine hotspots images, to provide equal weighting for each mouse, using inForm analysis software (PerkinElmer).
6
Multispectral Imaging and Cell Phenotyping
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Methodology adapted from 16 (link). Slides were scanned using 20x objectives with the PerkinElmer Vectra 3 automated multispectral imaging system. Images were imported into InForm analysis software (PerkinElmer), and analysis was performed according to manufacturer instructions and methods adapted from 16 (link). Cells were phenotyped based on one or multiple markers (E-cadherin only, KRT8 & E-cadherin, KRT14 only, Triple positive (KRT8+E-cadherin+ Vimentin), Snail only, Vimentin only, and Vimentin+ZEB1) and validated by marker distribution. Entire Cell Mean Fluorescent units were extracted for each marker and normalized as a percentile of maximum and minimum fluorescence across all cells in all images.
7
Quantifying Tumor-Infiltrating Lymphocytes
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Slides were imaged at 4 × and 20 × magnification using an Eclipse 80i microscope (Nikon). Infiltrating CD3+, CD4+, CD8+ and LAG3+ cells were enumerated from 40× fields using inForm analysis software (Perkin-Elmer). The density of staining in infiltrating T cells was calculated by the number of positive cells/mm2 as previously described27 (link),28 (link). The positively stained cells were quantified independently by two pathologists.
8
Tissue Imaging and Cell Phenotyping
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Whole-slide scans were captured at 10× with the PerkinElmer Vectra3 Slide Scanner, and ~50 ROIs per tumor were chosen manually with PhenoChart (PerkinElmer). ROIs were imaged at 20× resolution and imported into InForm analysis software (PerkinElmer). Spectral unmixing single stains and background fluorescence slides were generated from the parental tumor according to the OPAL Assay Development Guide. ROIs were spectrally unmixed and assigned colors and exported as composite images (Fig. 4A ). Tissue segmentation (trainable to 98% accuracy) and cell segmentation were performed (nuclear compartment —DAPI; cytoplasm—vimentin and KRT8; and membrane—E-cadherin), and cells were phenotyped on the basis of expression of one or multiple markers [E-cadherin only, KRT8/14 and E-cadherin, KRT8 and/or KRT14, triple positive (KRT8 + E-cadherin + vimentin), KRT8/14 and vimentin, Snail only, vimentin only, and vimentin + ZEB1] and validated by marker distribution (fig. S5, A and B). Entire cell mean fluorescent units were extracted for each marker and normalized as a percentile of maximum and minimum fluorescence across all cells in all images.
9
EGFR Expression in Glioblastoma Samples
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Tissue microarrays containing duplicates of 33 glioblastoma cases, 2 brain anaplastic astrocytoma, and 5 normal brain tissue samples were ordered from US Biomax, Inc. (Cat: GL806c). Arrays were stained with primary mouse anti-human EGFR antibody (Clone 31G7, Invitrogen) at a 1:100 dilution. Briefly, slides were baked for 30 minutes at 60°C and deparaffinized on the Leica Bond Automated Immunostainer, followed by antigen retrieval with Proteinase K for 10min at 37°C. Blocking was performed for 10min at RT using Normal Goat Serum (10% in TBS) followed by primary antibody in Leica primary antibody diluent for 30min. Secondary antibodies (Life Technologies) were incubated with TMAs for 2hr at RT and diluted 1:500 in PBS with 0.2% BSA.
TMAs were imaged using the Nuance Multispectral imaging system (Perkin Elmer) on a Nikon Eclipse Ci upright microscope at 20 ×. Images were captured every 20 nm wavelength of light from 420 nm-720 nm. Data were analyzed by InForm analysis software (Perkin Elmer) using a threshold of 0.07OD.
TMAs were imaged using the Nuance Multispectral imaging system (Perkin Elmer) on a Nikon Eclipse Ci upright microscope at 20 ×. Images were captured every 20 nm wavelength of light from 420 nm-720 nm. Data were analyzed by InForm analysis software (Perkin Elmer) using a threshold of 0.07OD.
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