Immunofluorescence Technique to Detect Subcellular Structures Critical to Oocyte Maturation

Abstract

Immunofluorescence is a useful technique for analysis of protein expression and localization, thereby providing information regarding protein function, regulation, and protein–protein interactions. It is a standard approach to determine the temporal and spatial location of gene products that function in oocyte meiotic maturation. Fixation is one of the critical steps in the immunofluorescence protocol. Here, we describe the use of antibodies that are widely utilized in oocytes studies: anti-centromeric antigen (ACA), anti-Aurora kinase A (AURKA) and anti-alpha and gamma-tubulin antibodies that require different technical approaches for successful visualization, and we provide protocols for these conditions that are amenable to mouse oocyte studies. Detection of these proteins provides phenotypic information about spindle morphology, chromosome alignment, and microtubule attachments to kinetochores critical to assessing oocyte quality.