Drosophila devitellinization

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. 

In addition, some Drosophila mutations that affect vitellogenesis result in embryos that cannot be devitellinized after formaldehyde fixation. The MeOH/EGTA procedure can bypass this problem. 

Formaldehyde Fixation of Drosophila Embryos Followed by Cold MeOH Devitellinization... 

One-Step MeOH Fixation/Devitellinization of Drosophila Embryos... 

Figure 9.4. Confocal micrographs of wild-type Drosophila embryos. (A) Whole-mount Drosophila embryos stained with propidium iodide to visuahze the nuclear division cycle. Shown are syncytial embryos in nuclear division cycles 1-13 and interphase of nuclear cycle 14 (cellularization). Bar, 100 pm. (B) Surface views of Drosophila embryos prepared by hand devitellinization and double-stained for actin (fluorescein phalloidin, green) and DNA (propidium iodide, red). Interphase actin caps (top panel) and actin-based metaphase furrows middle panel) are shown for embryos in nuclear division cycle 13. Bottom panel) Embryo at cellularization. Actin-based cellularization furrows surround each of the nuclei. Bar, 10 pm. (C) Surface view of a Drosophila embryo in late metaphase/early anaphase of nuclear division cycle 13. The embryo was prepared by hand devitellinization and triple-stained for actin (fluorescein phaUoidin,green), DNA (propidium iodide,pi/rp/e), and the furrow component, Dah (anti-Dah, Cy5-labeled secondary, red). Both actin and Dah locaUze to the furrows and appear to colocalize in some regions yellow staining, inset). Bar, 10 pm. Inset) 2X magnification.

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See also in sourсe #XX -- [ , ]

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