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Protocol for Efficient Protein Synthesis Detection by Click Chemistry in Colorectal Cancer Patient-Derived Organoids Grown In Vitro.

ABSTRACT: Here, we describe a protocol to detect and visualize protein synthesis by click-chemistry-based immunofluorescence in patient-derived organoids (PDOs) in vitro. The protocol uses O-propargyl puromycin (OPP), an analog of puromycin that enters the acceptor site of ribosomes and is incorporated into nascent polypeptides. OPP can be detected by a click chemistry reaction and can be combined with conventional antibody staining. We describe procedures for imaging intact organoids in 3D format or imaging sections of organoids from paraffin blocks. For complete details on the use and execution of this protocol, please refer to Morral, Stanisavljevic et al. (2020).

SUBMITTER: Morral C

PROVIDER: S-EPMC7501725 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Protocol for Efficient Protein Synthesis Detection by Click Chemistry in Colorectal Cancer Patient-Derived Organoids Grown <i>In Vitro</i>.

Morral Clara C Stanisavljevic Jelena J Batlle Eduard E

STAR protocols 20200918 2

Here, we describe a protocol to detect and visualize protein synthesis by click-chemistry-based immunofluorescence in patient-derived organoids (PDOs) <i>in vitro</i>. The protocol uses O-propargyl puromycin (OPP), an analog of puromycin that enters the acceptor site of ribosomes and is incorporated into nascent polypeptides. OPP can be detected by a click chemistry reaction and can be combined with conventional antibody staining. We describe procedures for imaging intact organoids in 3D format ...[more]

PMID: 33000005

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