Multiplex Immunofluorescence Histology Staining, Imaging and Analysis

The MITS Core offers comprehensive services to run 7-color multiplex immunofluorescence histology (mIFH) staining, imaging and analyses on paraffin-embedded human or murine tissue specimens. These services include panel development, slide staining, image acquisition with the Vectra microscope, region of interest selection, and quantitative analyses of images by digital software for cell enumeration data, marker expression and more.

Panels are composed of up to six different cell markers plus a nuclear stain (DAPI) to be imaged simultaneously on a single tissue specimen/slide.  This technique is useful for investigators who want to identify and enumerate cell subsets that express single or multiple antigenic markers or would like to understand the location and spatial relationship of cells to one another. The OPAL dyes utilized in this staining technique are useful for amplifying signal from weaker antigen targets, an advantage over traditional IHC. Additionally, the fluorescent dyes are stable allowing image analysis of tissue specimens to be sustained for at least a year.

The Vectra3 imaging system is comprised of a microscope and robot arm that enables high throughput image acquisition from up to 200 slides stained with traditional or fluorescent IHC techniques. Analyses to enumerate cell subsets or to perform spatial analyses can be done with Halo Software.

Available ready-to-use immunofluorescence histology panels are listed below, or you may contact Ileana Mauldin for assistance with development of a custom 7-color panel to meet your specific research needs.

mIFH Panels for human FFPE tissue specimens:

• T cell activation panel: CD4 or CD4, CD8, CD45RO, Grzm B, ICOS1, Ki67, DAPI
• Myeloid panel: CD11b, CD1a, GAL9, CD68, CD83, CD40, DAPI
• Checkpoint panel 1: PDL1, CD8, CD56, IFNg, SOX10/CK (pan-cytokeratin), DAPI
• Checkpoint panel 2: GAL9, LAG3, CD8, TIM3, CD155, MHCII
• Immune suppression panel: CD34, IDO1, ARG1, CD39, CD73, DAPI
• IL-10 panel: CD3 or CD4, CD8, CD20, CD34, T-bet, IL-10, DAPI
• T-helper lineage panel: CD3 or CD4, CD8, FoxP3, Tbet, GATA3, RORgt, DAPI
• Th panel: CD3 or CD4, CD20, FoxP3, Tbet, GATA3, RORgt, DAPI
• Ab production panel: CD3 or CD4, CD8, CD20, IFNg, IgG, Ki67, DAPI
• Plasma cell panel: CD or CD4, CD8, CD20, IgG, CD138, Ki67, DAPI
• Specific Investigator panel: 1 PD1/BLIMP/TIM3, CD8, GrzmB, IFNg, T-bet, Ki67, DAPI
• TLS panel 1: CD3 or CD4, CD8, CD20, PNAd, DCLamp, Ki67
• TLS panel 2: MADCAM, CD8, CD20, PNAd, RORgt, OLIG2, DAPI
• Immunotype panel*: CD8, CD45RO, CD34, Tbet, and Sox10 or Pancytokeratin and can have CD3 put it (Sox10 to identify melanoma cells; pan-cytokeratin to distinguish a number of cancer cells).
• Additional markers can be substituted in: CD34, CD45RA, RORgt, SIRPa

mIFH Panels for murine FFPE tissue specimens:

• Murine TLS: CD4, CD8, CD19, CD11c, PNAd, CD34, DAPI
• Murine T cell Activity: CD4, CD8, CD19, Granzyme-b, CD34, Ki67, DAPI

*Panels can be customized or created for your study’s needs

For questions regarding Multiplex Immunofluorescence histology Services, please contact: Dr. Ileana Mauldin at 434-982-3508 or is3v@virginia.edu.

Phase I/II clinical trial of a helper peptide vaccine plus PD-1 blockade in PD-1 antibody-naïve and PD-1 antibody-experienced patients with melanoma (MEL64). Vavolizza RD, Petroni GR, Mauldin IS, Chianese-Bullock KA, Olson WC, Smith KT, Dengel LT, Haden K, Grosh WW, Kaur V, Varhegyi N, Gaughan EM, Slingluff CL Jr. J Immunother Cancer. 2022 Sep; 10(9):e005424. DOI: 10.1136/jitc-2022-005424. PMID: 36100309.

Proliferating CD8+ T Cell Infiltrates Are Associated with Improved Survival in Glioblastoma. Mauldin IS, Jo J, Wages NA, Yogendran LV, Mahmutovic A, Young SJ, Lopes MB, Slingluff CL Jr, Erickson LD, Fadul CE. Cells. Dec 1; 10(12):3378. 2021. DOI: 10.3390/cells10123378.

Immune Mechanisms Orchestrate Tertiary Lymphoid Structures in Tumors via Cancer-Associated Fibroblasts. Rodriguez AB, Peske JD, Woods AN, Leick KM, Mauldin IS, Meneveau MO, Young SJ, Lindsay RS, Melssen MM, Cyranowski S, Parriott G, Conaway MR, Fu YX, Slingluff CL Jr, Engelhard VH. Cell Rep. Jul 20; 36(3):109422, 2021. DOI: 10.1016/j.celrep.2021.109422.

Heterogeneity in tertiary lymphoid structure B-cells correlates with patient survival in metastatic melanoma. Lynch KT, Young SJ, Meneveau MO, Wages NA, Engelhard VH, Slingluff CL Jr, Mauldin IS. J Immunother Cancer. Jun; 9(6):e002273, 2021. DOI: 10.1136/jitc-2020-002273.

Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Structures. Mauldin IS, Mahmutovic A, Young SJ, Slingluff CL Jr. Methods Mol Biol. 2021; 2265:573-587. 2021. DOI: 10.1007/978-1-0716-1205-7_40.

Immunotyping and Quantification of Melanoma Tumor-Infiltrating Lymphocytes. Max O Meneveau, Zeyad T Sahli, Kevin T Lynch, Ileana S Mauldin, Craig L Slingluff Jr. Methods Mol Biol. 2265:515-528. 2021. DOI: 10.1007/978-1-0716-1205-7_36.