Work

Protocol for identifying protein synthesis activity in specific cell types of the testicular lumen

Year: 2025

Type: article

Abstract: Protein synthesis could control spermatogenic cell fate transitions. Here, we present a protocol for visualization and quantification of newly synthesized proteins by click-chemistry-based immunofluorescence within specific spermatogenic cell types in the mice testicular lumen. We detail the processes for O-propargyl-puromycin (OPP) incorporation, antibody incubation, confocal microscope imaging, and subsequent quantification methods. This protocol is not limited to spermatogenic cells and can be adapted to investigate protein synthesis in other testicular cell types and various tissue-specific cell populations. For complete details on the use and execution of this protocol, please refer to Zou et al. 1 • Protocol for visualizing protein synthesis • Click chemistry combined with cellular immunofluorescence • Analysis of protein synthesis levels in spermatogenic cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Protein synthesis could control spermatogenic cell fate transitions. Here, we present a protocol for visualization and quantification of newly synthesized proteins by click-chemistry-based immunofluorescence within specific spermatogenic cell types in the mice testicular lumen. We detail the processes for O-propargyl-puromycin (OPP) incorporation, antibody incubation, confocal microscope imaging, and subsequent quantification methods. This protocol is not limited to spermatogenic cells and can be adapted to investigate protein synthesis in other testicular cell types and various tissue-specific cell populations.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.103661

Authors: Dingfeng Zou, Kai Li, Yan Lu, Wei Yan, Wei Song

Institutions: Chinese Academy of Medical Sciences & Peking Union Medical College, Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, University of California, Los Angeles, The Lundquist Institute, Harbor–UCLA Medical Center

Language: English

Topic: Sperm and Testicular Function

Subfield: Reproductive Medicine

Field: Medicine

Domain: Health Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funders: National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences and Peking Union Medical College, National Key Research and Development Program of China