Multiplex Immunofluorescence Staining and Analysis

Sectioning and Staining: After harvesting, tumors were immediately fixed overnight in 10% neutral-buffered formalin. Fixed tumors were embedded in paraffin and sections were cut at a thickness of 5 μm. Full-section slides of tumor tissues were stained using Opal multiplex 6-plex kits, according to the manufacturer’s protocol (PerkinElmer), for DAPI, Epcam (polyclonal; Abcam, 1:100 dilution), CD3 (clone SP7; Spring Biosciences; 1:100 dilution), CD8 (clone 4SM15; ThermoFisher; 1:500), CD4 (clone 4SM95; eBioscience, 1:50), Foxp3 (polyclonal; ThermoFisher, 1:500), and Granzyme B (polyclonal; Abcam, 1:200). Single color controls and an unstained slide were also included.
Multispectral imaging and analysis: Multispectral image capture was done at 20X magnification using Vectra (PerkinElmer, Hopkinton, MA). Images were analyzed using inForm software version 2.4.1 (PerkinElmer) as previously described [31 (link)]. Further details are presented in the Supplementary Materials.

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Hanoteau A., Newton J.M., Krupar R., Huang C., Liu H.C., Gaspero A., Gartrell R.D., Saenger Y.M., Hart T.D., Santegoets S.J., Laoui D., Spanos C., Parikh F., Jayaraman P., Zhang B., Van der Burg S.H., Van Ginderachter J.A., Melief C.J, & Sikora A.G. (2019). Tumor microenvironment modulation enhances immunologic benefit of chemoradiotherapy. Journal for Immunotherapy of Cancer, 7, 10.

Publication 2019

Opal multiplex 6-plex kits Epcam CD8 (clone 4SM15; CD4 (clone 4SM95; Granzyme B inForm software version 2.4.1

Clone Dapi Dilution Embedded paraffin Epcam Formalin Granzyme b Opal Tissues Tumor Vectra

Corresponding Organization :

Other organizations : Baylor College of Medicine, Center for Translational Molecular Medicine, University of Lübeck, Research Center Borstel - Leibniz Lung Center, University Hospital Schleswig-Holstein, Breast Center, Texas Health Dallas, Penn Presbyterian Medical Center, Pediatric Oncology Group, Columbia University Irving Medical Center, Leiden University Medical Center, Vrije Universiteit Brussel, VIB-UGent Center for Inflammation Research, University of South Dakota, ISA Pharmaceuticals (Netherlands)

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of DAPI, Epcam, CD3, CD8, CD4, Foxp3, and Granzyme B

control variables

Tumor tissue fixed overnight in 10% neutral-buffered formalin
Tumor tissue embedded in paraffin and sectioned at 5 μm thickness
Staining performed using Opal multiplex 6-plex kits according to manufacturer's protocol
Multispectral imaging performed at 20X magnification using Vectra
Image analysis using inForm software version 2.4.1

controls

Single color controls
Unstained slide

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