The Importance of Adequate Fixation for Immunofluorescent Staining of Bovine Embryos

Authors

  • K Goossens,

    1. Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • L Vandaele,

    1. Reproductive Biology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • E Wydooghe,

    1. Reproductive Biology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • M Thys,

    1. Reproductive Biology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • J Dewulf,

    1. Reproductive Biology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • LJ Peelman,

    1. Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author
  • A Van Soom

    1. Reproductive Biology Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
    Search for more papers by this author

Author’s address (for correspondence): K Goossens, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. E-mail: karen.goossens@ugent.be

Contents

Immunofluorescent staining is often used to investigate the expression of specific proteins in pre-implantation embryos. The success of this method is determined by the specificity of the antibodies, but also by the protocol used for fixation and permeabilization of the samples. In this study, different fixatives are compared in combination with immunofluorescent staining of caudal-type homeobox 2 (CDX2), fibronectin 1 (FN1) and integrins (ITGs) on bovine blastocysts. For both CDX2 and the ITGs, the outcome of the staining was largely dependent on the fixation methods. Paraformaldehyde fixation was best for the intracellular CDX2 protein, whereas acetone fixation gave the best results for the transmembrane ITGs. No difference was observed for the FN1 staining between samples fixed with paraformaldehyde or acetone. These examples demonstrate that the choice of fixation and permeabilization agents is very important for the outcome of the experiment, and this choice is dictated by the (extra)cellular location of the protein under investigation. Inappropriate fixation and/or permeabilization methods can lead to erroneous conclusions regarding the site and amount of protein expression.

Ancillary