NanoString spatial profiling was used for DSP analysis of three protein-level and 1800 transcriptomic-level immune-markers simultaneously on FFPE tissue slides45 (link), 46 (link). Following deparaffinization and antigen retrieval procedures, sections were simultaneously incubated overnight with fluorescent-labeled antibodies against CD3 (Origene), CD68 (Santa Cruz) and Pan-Cytokeratin (Novus Biologicals), smooth muscle actin (Invitrogen), CD20 (Novus Biologicals), or cytokeratin 8/18 (Novus Biologicals) to visualize morphological features in the regions of interest (ROI). After staining, the tissues were scanned using a GeoMx DSP instrument to generate digital fluorescent images and to select for individual ROIs inside a geometric section. To carry out high-resolution multiplex profiling, each ROI was assigned to CD3+ T-cell-rich or CD68+ macrophage-rich regions. UV light was directed through a programmable digital micro mirror device (DMD) or dual DMD (DDMD) to accurately illumine the ROI and cleave PC oligos from the selected region, which were collected by microcapillary tube inspiration and dispensed into a 96-well plate. Inside the microplate, the single-molecule counting nCounter System enabled the digital counting of released oligos47 (link), 48 (link).
Pan cytokeratin
Manufactured by Novus Biologicals
Pan-Cytokeratin is a cocktail of monoclonal antibodies that collectively recognize a broad spectrum of cytokeratin subtypes. Cytokeratins are intermediate filament proteins found in the intracytoplasmic cytoskeleton of epithelial cells. This antibody can be used to identify a wide range of normal and neoplastic epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Human osteoactivin gpnmb anti goat, by R&D Systems (1 mentions)
Melan a, by Abcam (1 mentions)
Cytokeratin 7, by Abcam (1 mentions)
Tfe3 anti rabbit, by Merck Group (1 mentions)
Cathepsin k, by Abcam (1 mentions)
Axio scan z1 slide scanner, by Zeiss (1 mentions)
F4 80, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions)
Inform advanced image analysis software, by Akoya Biosciences (1 mentions)
Cd11b, by Abcam (1 mentions)
3 protocols using pan cytokeratin
1
Spatially Resolved Protein and Transcriptomic Profiling
2
Immunohistochemical Profiling of Tumor Samples
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Five-micron thick formalin-fixed paraffin-embedded sections were cut from all available tumor blocks. IHC staining and H&E staining were performed by either VitroVivo Biotech (Rockville, MD) or the Molecular Histopathology Laboratory at the Frederick National Laboratory for Cancer Research (Frederick, MD) using standardized methodologies. IHC staining was performed using either Human Osteoactivin/GPNMB anti-goat (R&D Systems, Minneapolis, MN), TFE3 anti-rabbit (Sigma Aldrich, Burlington, MA), Phospho-S6 Ribosomal Protein (Ser235/236) anti-rabbit (Cell Signaling Technology, Danvers, MA), or Phospho-4-E-BP1 (Thr37/46) anti-rabbit (Cell Signaling Technology, Danvers, MA). For selected tumors, an immunohistology panel was performed evaluating HMB45 (Agilent Dako, Carpinteria, CA), Melan-A (Abcam, Waltham, MA), Pan-cytokeratin (Novus Biologicals, Centennial, CO), Cytokeratin 7 (Abcam, Waltham, MA), Cathepsin K (Abcam, Waltham, MA), and Carbonic anhydrase IX (Cell Signaling Technology, Danvers, MA). Images were captured using an AxioScan.Z1 Slide Scanner (Zeiss, Oberkochen, DE).
3
Multiplex Immunohistochemistry for Immune Cell Profiling
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mIHC staining was performed according to the manufacturer’s instructions (Opal™ 4-Color Anti-Rabbit Automation IHC; Opal™ Polymer Anti-Rabbit HRP Secondary Antibody Kit; Akoya Biosciences®). The following primary rabbit antibodies were used for staining: CD4 (CST25229; 1:200), CD8 (CST98941; 1:400), F4/80 (CST 70076; 1:1000), and Ly6G (CST 87048; 1:1000) were purchased from Cell Signaling Technology®; CD11b (Ab133357; 1:3000) from Abcam®; PanCytokeratin (NBP3–07280; 1:1000) from Novus Biologicals®. Nuclei were stained with DAPI (Akoya Biosciences®). Slides were scanned using the “Vectra Polaris” platform (Akoya Biosciences®). Spectral unmixing and further tissue /cell segmentation as well as phenotyping were performed using the inForm Advanced Image Analysis software (inForm v2.4.10, Akoya Biosciences®). Multiplex data analyses: Data output from inForm 2.4.10 was further processed in RStudio (Rstudio v1.1.456) using “R” packages “Phenoptr” and “PhenoptrReports” with R version 4.1.0. Consolidated results for “Cell densities” were exported and used for further analyses.
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