Multiplex Immunofluorescence Staining and Analysis

The multispectral immunofluorescence (IF) staining method was performed as previously reported [38 (link)]. The samples were stained using the following antibodies: anti-CD8 (ZCIA055, zuochengbio), anti-CD57 (ZCIA273, zuochengbio), anti-GZMA (ab209205, Abcam), and anti-GSDMB (12885-1-AP, ThermoFisher). The samples were mounted using ProLong Diamond Antifade mounting medium containing DAPI (Invitrogen) and then sliced into 5 μm sections before being placed onto adhesive microscope slides. We performed deparaffinization, rehydration, and antigen retrieval to prepare the slides for multiplexed immunofluorescence staining. TG TSA Multiplex IHC Assay Kits (TissueGnostics Asia-Pacific Ltd.) were used along with a spectral library constructed from single-staining tissue images of each reagent to guide the staining process. The imaging procedure was performed utilizing the TissueFAXS (TissueGnostics) system, in conjunction with the Zeiss Axio Imager Z2 Microscope System, at a magnification of ×20. Quantitative analysis of the stained samples involved measuring cell density, expression levels, and area per cell using StrataQuest software (version 7.1.129, TissueGnostics GmbH, Vienna, Austria).

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Zhang Y., Bai Y., Ma X.X., Song J.K., Luo Y., Fei X.Y., Ru Y., Luo Y., Jiang J.S., Zhang Z., Yang D., Xue T.T., Zhang H.P., Liu T.Y., Xiang Y.W., Kuai L., Liu Y.Q, & Li B. (2023). Clinical-mediated discovery of pyroptosis in CD8+ T cell and NK cell reveals melanoma heterogeneity by single-cell and bulk sequence. Cell Death & Disease, 14(8), 553.

Publication 2023

ab209205 ProLong Diamond Antifade mounting medium TG TSA Multiplex IHC Assay Kits TissueFAXS Axio Imager Z2 Microscope System StrataQuest software

Antibodies Antigen Assay Cell Dapi Diamond Gzma Immunofluorescence Library Microscope Rehydration Staining tissue

Corresponding Organization :

Other organizations : Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai Skin Disease Hospital, Tongji University

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Variable analysis

independent variables

Anti-CD8 antibody (ZCIA055, zuochengbio)
Anti-CD57 antibody (ZCIA273, zuochengbio)
Anti-GZMA antibody (ab209205, Abcam)
Anti-GSDMB antibody (12885-1-AP, ThermoFisher)

dependent variables

Cell density
Expression levels
Area per cell

control variables

Deparaffinization, rehydration, and antigen retrieval procedures
TG TSA Multiplex IHC Assay Kits (TissueGnostics Asia-Pacific Ltd.)
Spectral library constructed from single-staining tissue images
Imaging procedure using TissueFAXS (TissueGnostics) system and Zeiss Axio Imager Z2 Microscope System at ×20 magnification
Quantitative analysis using StrataQuest software (version 7.1.129, TissueGnostics GmbH, Vienna, Austria)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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