Work

Protocol for simultaneous detection of mRNAs and (phospho-)proteins with ARTseq-FISH in mouse embryonic stem cells

Year: 2024

Type: article

Abstract: Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibo'dies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments. For complete details on the use and execution of this protocol, please refer to Hu et al. 1 • Guidelines for probe design and instructions for antibody conjugation • Optimized protocol for combining the hybridization of selected mRNAs and proteins • Image-based multiplexed detection of mRNAs and (phospho-)proteins in single cells • Application of ARTseq-FISH for other cell lines and detection approaches (FACS) Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibodies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103336

Authors: Xinyu Hu, Bob van Sluijs, Óscar García-Blay, Wilhelm T.S. Huck, Maike M.K. Hansen

Institutions: Radboud University Nijmegen, Oncode Institute

Language: English

Topic: Single-cell and spatial transcriptomics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,500