Spanish Institute of Oceanography (IEO)

1 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

David G. Valcarce^1,2, Vanesa Robles^2,3

¹Department of Molecular Biology, University of León, ²INDEGSAL, University of León, ³Planta de Cultivos el Bocal, Spanish Institute of Oceanography (IEO)

This protocol describes a detailed methodology for detecting H₂O₂ localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.