Abstract
Formation of full-grown oocytes requires the control and coordination of a number of processes (e.g., oocyte growth) through multiple stages, where disruption at any one step can result in infertility. Numerous proteins are required for the regulation and execution of the various oogenic processes as well as functioning as maternal products needed for embryogenesis. Immunofluorescence microscopy combined with staining using antibodies against specific proteins, or their posttranslationally modified forms, is a standard approach to determine the temporal and spatial location of gene products that function in oocyte development. The simple linear organization of the germline in the model organism Caenorhabditis elegans allows easy correlation of protein localization and germ cell developmental stage, thus aiding in our understanding of protein function during gametogenesis. Here we outline co-immunofluorescence staining for two major regulators of C. elegans germline development, the translational repressor GLD-1 and activated form of MPK-1 (dpMPK-1) ERK MAP kinase in dissected gonads from adult C. elegans. Worms are first dissected and the extruded gonads are fixed and permeabilized before being bathed in primary antibodies against GLD-1 and dpMPK-1. Secondary antibodies conjugated to fluorophore dyes and that target the IgG domains of the primary antibody reagents are then used to provide a fluorescent signal that corresponds to the position of GLD-1 and dpMPK-1. The outlined procedure is amenable to many other proteins expressed in C. elegans germ cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Coons AH, Creech HJ, Jones RN (1941) Immunological properties of an antibody containing fluorescent group. Exp Biol Med 47:200–202
Coons AH, Kaplan MH (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 91:1–13
Francis R, Barton MK, Kimble J et al (1995) gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics 139:579–606
Hansen D, Hubbard EJA, Schedl T (2004) Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline. Dev Biol 268:342–357
Fox PM, Vought VE, Hanazawa M et al (2011) Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline. Development 138:2223–2234
Jones AR, Francis R, Schedl T (1996) GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenorhabditis elegans germline development. Dev Biol 180:165–183
Lee MH, Ohmachi M, Arur S et al (2007) Multiple functions and dynamic activation of MPK-1 extracellular signal-regulated kinase signaling in Caenorhabditis elegans germline development. Genetics 177:2039–2062
Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465
Acknowledgments
This work is supported by NIH F32GM106615 to JB and R01GM100756 to TS.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Brenner, J.L., Schedl, T. (2016). Indirect Immunofluorescence of Proteins in Oogenic Germ Cells of Caenorhabditis elegans . In: Nezis, I. (eds) Oogenesis. Methods in Molecular Biology, vol 1457. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3795-0_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3795-0_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3793-6
Online ISBN: 978-1-4939-3795-0
eBook Packages: Springer Protocols