Chapter 9 Confocal Immunofluorescence Microscopy of Microtubules in Amphibian Oocytes and Eggs

Abstract

Publisher Summary The advent and increasing availability of confocal microscopes has dramatically facilitated immunofluorescence microscopy of microtubule organization in large cells. The optical sectioning capabilities afforded by confocal microscopy eliminate the out-of-focus fluorescence that degrades conventional epifluorescence images of large cells and allows the rapid collection of images without the inconvenience of physical sectioning or complicated computerized deconvolution. This chapter focuses on the confocal immunofluorescence microscopy to examine microtubules organization in amphibian oocytes, eggs, and early embryos. The chapter provides a practical guide to immunofluorescence and confocal laser scanning microscopy (CLSM) of microtubules. The chapter compares the preservation of microtubules in oocytes fixed by several different protocols. Several problems were often encountered with cells fixed in methanol—significant shrinkage and distortion of oocytes, poor preservation of microtubules, and fragile oocytes. The most consistent preservation of individual microtubules throughout oogenesis and early embryogenesis was obtained with combinations of formaldehyde and glutaraldehyde. The chapter compares glutaraldehyde concentrations ranging from less than 0.1 to 2%.