Chapter 9 Confocal Immunofluorescence Microscopy of Microtubules in Oocytes, Eggs, and Embryos of Algae and Amphibians

Abstract

Publisher Summary This chapter discusses the experiences of using immunofluorescence combined with confocal laser-scanning microscopy (CLSM) to examine cytoskeletal organization in algal and amphibian oocytes, eggs, and early embryos. Preparation of Pelvetia or Xenopus eggs for immunofluorescence microscopy follows the same procedure as is used for more typical samples, such as cultured cells grown on coverslips. Many factors enter into the design or choice of a fixation protocol for immunofluorescence. However, two factors are of paramount importance—namely, (1) the fixation protocol must be compatible with the antibodies or staining technique to be used and (2) the fixation procedure must adequately immobilize and preserve the antigen under study and the cellular structures with which it is associated. Heavy deposits of pigment in the cortex and cytoplasm of amphibian oocytes and eggs hamper immunofluorescence microscopy of these cells. Dent and Klymkowsky (1989) demonstrated that a solution of hydrogen peroxide in methanol could be used to bleach Xenopus eggs, eliminating the interfering pigment. Extended bleaching of Xenopus oocytes (up to 72 hr) does not appear to affect either the structural preservation or reactivity of microtubules with tubulin antibodies. Autofluorescence resulting from the use of glutaraldehyde-containing fixatives can be minimized by treating cells with sodium borohydride prior to incubating with antibodies.