Multiplex Immunohistochemistry for Tissue Analysis

Tissue sections were prepared from formalin-fixed paraffin embedded tissue blocks and cut to 4 μm serial sections and mounted on Superfrost Plus (VWR). The procedure for multiplex immunohistochemistry (mIHC) was followed by a manufacturer’s protocol for Opal7-color automation IHC kit (Akoya Bioscience), and the staining was performed with Autostainer DISCOVERY ULTRA (Ventana). Antibodies used in mIHC are anti-CD3 (clone 2GV6, Ventana), anti-CD20 (clone L26, Ventana), anti-Ki67 (clone 30-9, Ventana), anti-FOXP3 (clone SP97, Spring), anti-pan cytokeratin (CK; clone AE1/AE3, DAKO), anti-CD117 (clone c-kit, DAKO). The molecular markers of immune panel (CD3, CD20, Ki67, CKs, FOXP3, and CD117) were visualized with Opal520, Opal540, Opal570, Opal620, Opal650, and Opal690, respectively. DAPI counterstaining was performed with Discovery QD DAPI (Roche). ProLong Diamond Antifade Mounting (ThermoScientific) was used for mounting the coverslip. Detailed staining conditions and autostainer’s protocols are reported in our recent report^{86 (link)}.

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Nachmanson D., Officer A., Mori H., Gordon J., Evans M.F., Steward J., Yao H., O’Keefe T., Hasteh F., Stein G.S., Jepsen K., Weaver D.L., Hirst G.L., Sprague B.L., Esserman L.J., Borowsky A.D., Stein J.L, & Harismendy O. (2022). The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ. NPJ Breast Cancer, 8, 6.

Publication 2022

Superfrost Plus Opal7-color automation IHC kit anti-CD3 (clone 2GV6, anti-CD20 (clone L26, anti-Ki67 (clone 30-9, ProLong Diamond Antifade Mounting

Anti cd3 Antibodies Clone Cytokeratin Dapi Diamond Embedded paraffin Formalin Immunohistochemistry Molecular markers Tissue

Corresponding Organization :

Other organizations : University of California, San Diego, University of California, Davis, University of Vermont, UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco

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Variable analysis

independent variables

Tissue sections prepared from formalin-fixed paraffin embedded tissue blocks
Multiplex immunohistochemistry (mIHC) procedure followed by manufacturer's protocol for Opal7-color automation IHC kit (Akoya Bioscience)
Staining performed with Autostainer DISCOVERY ULTRA (Ventana)

dependent variables

Visualization of molecular markers of immune panel (CD3, CD20, Ki67, CKs, FOXP3, and CD117) with Opal520, Opal540, Opal570, Opal620, Opal650, and Opal690, respectively

control variables

Tissue sections cut to 4 μm serial sections and mounted on Superfrost Plus (VWR)
DAPI counterstaining performed with Discovery QD DAPI (Roche)
ProLong Diamond Antifade Mounting (ThermoScientific) used for mounting the coverslip

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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