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

Summary 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).
Graphical Abstract
Highlights • Protocol allows for protein synthesis visualization in CRC organoids by OPP incorporation • Protein synthesis can be observed within intact organoids in 3D, or in paraffin sections • OPP detection can be combined with antibody staining
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.