Multiplex Immunofluorescence Staining Protocol

Multiplex immunofluorescence staining was performed as previously described (20 (link), 21 (link)). The primary Abs were CD147 (Rabbit, 1:100, Proteintech, China), CD68 (Rabbit, 1:3000, AiFang biological, China), CD163 (Rabbit, 1:3000, Proteintech, China). The secondary antibody was horseradish peroxidase-conjugated secondary antibody incubation (GB23301, GB23303, Servicebio, China), and the tyramide signal amplification was TSA (FITC-TSA, CY3-TSA, 594-TSA, and 647-TSA (Servicebio, China)). Multispectral images were analyzed, and positive cells were quantified at a single-cell level by Caseviewer (C.V 2.3, C.V 2.0) and Pannoramic viewer (P.V 1.15.3) image analysis software. Negative control procedures included the omission of the primary antibody.

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Zhang J., Wang Z., Zhang X., Dai Z., Zhi-Peng W., Yu J., Peng Y., Wu W., Zhang N., Luo P., Zhang J., Liu Z., Feng S., Zhang H, & Cheng Q. (2022). Large-Scale Single-Cell and Bulk Sequencing Analyses Reveal the Prognostic Value and Immune Aspects of CD147 in Pan-Cancer. Frontiers in Immunology, 13, 810471.

Publication 2022

CD147 CD163 GB23301 GB23303 FITC-TSA CY3-TSA 594-TSA 647-TSA

Antibody Biological Cd147 Cd163 Cell Fitc Horseradish peroxidase Immunofluorescence Rabbit

Corresponding Organization : Xiangya Hospital Central South University

Other organizations : Affiliated Hospital of Guizhou Medical University, Guiyang Medical University, Harbin Medical University, Zhujiang Hospital, Southern Medical University, First Affiliated Hospital of Zhengzhou University

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Variable analysis

independent variables

Primary Abs: CD147 (Rabbit, 1:100, Proteintech, China), CD68 (Rabbit, 1:3000, AiFang biological, China), CD163 (Rabbit, 1:3000, Proteintech, China)

dependent variables

Positive cells quantified at a single-cell level by Caseviewer (C.V 2.3, C.V 2.0) and Pannoramic viewer (P.V 1.15.3) image analysis software

control variables

Negative control procedures included the omission of the primary antibody

controls

Negative control: Omission of the primary antibody

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