Protocol

Whole-Mount Preparation of Helobdella (Leech) Embryos for Microscopy

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA

¹Corresponding author (weisblat{at}berkeley.edu)

This article is also available in Emerging Model Organisms: A Laboratory Manual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

Due to the high yolk content and relatively large size of the Helobdella (leech) embryo, it is preferable to examine the embryos in a cleared whole-mount preparation after they have been stained. Three whole-mount procedures suitable for leech embryos are described here. The glycerol whole mount is quick and convenient because it does not require dehydration. However, prolonged incubation in buffered glycerol could cause the yolk to turn dark purple. Thus, it is not suitable for yolk-containing specimens that are intended for light microscopy. For fluorescence microscopy, anti-fading reagent can be added to the buffered glycerol. This article includes a recipe that works quite well for leech embryos. Also included are two alternative methods that require embryo dehydration, but have the advantage of clearing yolk-containing tissue. One method relies on the use of benzyl benzoate:benzyl alcohol (BBBA), a solution that provides the best clearing of yolk-containing tissue in the leech embryo. It is suitable for both fluorescence and light microscopy. Another alternative is the Epon whole-mount preparation. Epon is not quite as effective in clearing the yolk, but the viscosity of Epon facilitates orienting the specimens for imaging. As with BBBA, Epon is suitable for both light and fluorescence microscopy.