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Protocol for Efficient Protein Synthesis Detection by Click Chemistry in Colorectal Cancer Patient-Derived Organoids Grown In Vitro
- Source :
- STAR Protocols, Vol 1, Iss 2, Pp 100103-(2020), Star Protocols
- Publication Year :
- 2020
- Publisher :
- Elsevier, 2020.
-
Abstract
- 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).<br />Graphical Abstract<br />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<br />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.
- Subjects :
- Colorectal cancer
Cell Culture Techniques
Fluorescent Antibody Technique
Immunofluorescence
Ribosome
General Biochemistry, Genetics and Molecular Biology
chemistry.chemical_compound
Protocol
Protein biosynthesis
medicine
Organoid
Humans
lcsh:Science (General)
Staining and Labeling
General Immunology and Microbiology
medicine.diagnostic_test
General Neuroscience
Proteins
medicine.disease
Molecular biology
In vitro
Organoids
chemistry
Puromycin
Protein Biosynthesis
Click chemistry
Click Chemistry
Colorectal Neoplasms
Ribosomes
lcsh:Q1-390
Subjects
Details
- Language :
- English
- ISSN :
- 26661667
- Volume :
- 1
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- STAR Protocols
- Accession number :
- edsair.doi.dedup.....2e7d755bcb1d59f0ac6ab2ecca4b735a