Preparation of Fixed Drosophila Oocytes for Immunostaining: A High-Throughput Method to Fix and Remove the Outer Membrane

Abstract

Source: Radford, S. J., McKim, K. S. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J. Vis. Exp. (2016).

Immunostaining of Drosophila late-stage oocytes can prove challenging due to the oocytes' surrounding membranes. This video describes a high-throughput method for obtaining oocytes, their fixation, and membranes' removal for immunostaining. The featured protocol demonstrates these techniques with late-stage oocytes used to visualize different stages of meiosis.

Protocol

This protocol is excerpted from Radford and McKim, Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes, J. Vis. Exp. (2016).

NOTE: Procedures are performed at room temperature unless otherwise noted. Temperature-controlled incubators are used to maintain temperatures for fly rearing and crosses unless otherwise noted.

1. Preparations

1. Prepare Flies.
   1. Prometaphase-enriched oocyte collections.
      1. Clear adult flies from healthy, young stock or cross cultures. Age bottl....

Results

Drug	Solvent	Stock concentration	Final concentration	Time of treatment	Effect
colchicine	ethanol	125 mM	150 µM	10 min or 30 min	destabilize non-kinetochore (10 min) or all (.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% formaldehyde	Ted Pella, Inc.	18505	HAZARDOUS; once opened, discard after one month
250 ml beakers	Various
5 ml tubes	Various
active dry yeast	Various		mix with water to make a paste the consistency of peanut butter
Binucleine 2	Sigma	B1186	HAZARDOUS
blender	Various
bovine serum albumin	Sigma	A4161
calcium chloride	Various
colchicine	Sigma	C-9754	HAZARDOUS
coverslips	VWR	48366-227	No. 1 1/2
DMSO	Various
EGTA	Various
ethanol	Various
forceps	Ted Pella, Inc.	5622	Dumont tweezers high precision grade style 5
glass slides	VWR	48312-003
glucose	Various
HEPES	VWR	EM-5330	available from several venders
magnesium chloride	Various
large mesh (~1,500 µm)	VWR	AA43657-NK	variety of formats and other suppliers, 12 or 14 mesh
small mesh (~300 µm)	Spectrum labs	146 424	variety of formats, e.g., 146 422 or 146 486
nutator	Various
Pasteur pipets	Various
potassium acetate	Various
Cacodylic acid	Sigma	C0125	HAZARDOUS; alternatively, sodium cacodylate may be substituted
potassium hydroxide	Various
sodium acetate	Various
sodium hydroxide	Various
sucrose	Various
taxol (paclitaxel)	Sigma	T1912	HAZARDOUS
Triton X-100	Fisher	PI-28314
Tween 20	Fisher	PI-28320
vortex	Various