Multiplex Immunohistochemistry for Tumor-Infiltrating Lymphocytes

Paraffin-embedded tumor sections were deparaffinized, blocked, and stained with primary antibodies as described^{21 (link)}. Sequential staining was achieved with anti-mouse CD4 (clone 4SM95), Opal520 (PerkinElmer), CD8 (4SM15), and Opal620 with washes performed in Amplification Plus buffer (PerkinElmer). Nuclei counterstaining was achieved with Spectral DAPI. Slides were imaged on a Vectra Polaris Imager (PerkinElmer) and analyzed with QuPath software^{22 (link)}.

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Lee M.Y., Metenou S., Brough D.E., Sabzevari H., Bai K., Jochems C., Schlom J, & Allen C.T. (2021). Preclinical study of a novel therapeutic vaccine for recurrent respiratory papillomatosis. NPJ Vaccines, 6, 86.

Publication 2021

Opal520 Opal620

Antibodies Buffer Clone Dapi Mouse Nuclei Paraffin Tumor Vectra

Corresponding Organization :

Other organizations : National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Precigen (United States), National Cancer Institute

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Variable analysis

independent variables

Primary antibodies used for sequential staining

dependent variables

Immune cell populations (CD4+ and CD8+ T cells)

control variables

Paraffin-embedded tumor sections
Deparaffinization, blocking, and staining protocol
Washes performed in Amplification Plus buffer
Nuclei counterstaining with Spectral DAPI
Imaging on Vectra Polaris Imager
Analysis with QuPath software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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