Immunostaining embryos

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

Two protocols are provided for fixation of Drosophila embryos. Because these embryos are surrounded by both a chorion (eggshell) and an impermeable vitelline membrane, they require special steps to remove these barriers prior to immunostaining or in situ hybridization. Typically, the chorion is dissolved by treatment with sodium hypochlorite (commercial bleach) and the vitelline membrane is removed, either concomitant with or after fixation, by a combination of temperature and osmotic shock. The heptane in these procedures permits the fixative to pass through the vitelline membrane. 

Source or references Immunostaining embryos/larvae (dilutions) Westerns (dilutions) ECL Remarks... 

For Drosophila embryos, the best method to use is also the most common fixation procedure for immunostaining, as described by Rothwell and Sullivan (this volume see Protocol 9.3). Briefly, after bleach dechorionation, transfer embryos to a test tube or vial... 

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