Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging PMID: 31534232

RQC and Longevity Regulation

2026-09-03

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

Nat Protoc. 2019 Oct;14(10):2900-2930. doi: 10.1038/s41596-019-0206-y.

Ziming Du ^# ¹ ² , Jia-Ren Lin ^# ² ³ , Rumana Rashid ^# ¹ ⁴ , Zoltan Maliga ² ³ , Shu Wang ² ³ ⁵ , Jon C Aster ¹ ² , Benjamin Izar ² ³ ⁶ ⁷ , Peter K Sorger ⁸ ⁹ ¹⁰ , Sandro Santagata ¹¹ ¹² ¹³ ¹⁴ ¹⁵

Affiliations

1. Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
2. Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, USA.
3. Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA.
4. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.
5. Harvard Graduate Program in Biophysics, Harvard University, Boston, MA, USA.
6. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
7. Broad Institute of MIT and Harvard, Boston, MA, USA.
8. Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, USA. [email protected].
9. Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA. [email protected].
10. Department of Systems Biology, Harvard Medical School, Boston, MA, USA. [email protected].
11. Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. [email protected].
12. Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, USA. [email protected].
13. Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA. [email protected].
14. Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA, USA. [email protected].
15. Department of Pathology, Boston Children's Hospital, Boston, MA, USA. [email protected].
# Contributed equally.

Abstract

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, Macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.