Importance of cuticle preparations in Drosophila studies:
The Drosophila embryonic ventral epidermis has served as a unique tissue for the genetic analysis of patterning.
There are two different types of epidermal cells in the ventral epidermis which can be easily distinguished: those that secrete short, thick hair-like structures called denticles and cells that only secrete smooth cuticle.
Denticle-secreting cells form segmentally repeated belts.
This arrangement of epidermal cells in the denticle belts are due to influence of many signaling pathways during embryogenesis.
Cuticle patterns can be used as a sensitive readout of signaling activity and other patterning mechanisms.
For observing clear cuticle patterns, Hoyer’s medium is generally employed to clear internal tissues and leave intact cuticle.
Preparation of Hoyer’s medium:
30 g of Gum Arabica (Sigma-Aldrich, UK) was added to 50 mL of distilled water.
This was stirred overnight until completely dissolved, and then gradually 200 g of chloral hydrate (anhydrous) was mixed. To that 20 g of glycerol was added.
This mix needs to be centrifuged for 2 h at 25,000g.
The medium is stable for several years at room temperature. If the medium becomes too viscous
over the years, it can be diluted with water.
The addition of lactic acid to Hoyer’s-based medium increases the contrast in preparation and reduces clearing time. The 1:1 (v/v) mixture of Hoyer’s based medium and lactic acid is referred to here as Hoyer’s-based mountant. This
solution will digest all internal tissues but will leave the cuticle intact.
Cuticle preparations for embryos or first instar larvae:
Embryos needs to be collected after overnight laying at appropriate temperatures.
Later, the collected embryos will be processed to remove their chorion membrane using sodium hypochlorite or bleach.
The dechorinated embryos will be washed washed with tap water.
The desired genotypes should be transferred to a clean slide with sufficient amounts of 1:1 lactic acid: Hoyer’s-based medium and covered by a cover slip over the embryos or first instar larvae.
Using a forceps, the vitelline membranes of embryos needs to be broken by gently pushing the cover slip.
The slides should be then left on hot plate for few hours or overnight.
The slides can then be viewed under microscope.
Image Credit : wikipedia