Immunofluorescence Staining of K-Fibers in Mouse Oocytes Using Cold Fixation

The kinetochore is a multiprotein complex that assembles on centromeric DNA and constitutes the main attachment interface between chromosomes and microtubules of the spindle apparatus. Kinetochores also provide the platform for integrating the surveillance mechanism known as the spindle assembly checkpoint (SAC) that regulates the timing of anaphase onset. Saturation of microtubule binding sites on kinetochores displaces SAC proteins leading to loss of SAC-mediated inhibition and the triggering of anaphase. Microtubule binding sites become saturated by bundles of microtubules attached in an end-on manner to kinetochores, termed kinetochore fibers or K-fibers. The appearance of K-fibers therefore signifies the completion of attachment between kinetochores and microtubules and the silencing of the SAC. Here we describe a method involving cold-fixation for immunostaining and imaging K-fibers during meiosis I in mouse oocytes.