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Skin
organ culture. Fertile chicken eggs were grown in a
humidified incubator 38oC
for 7 and 8 days. Explants of dorsal skin were dissected
from the embryos in Howard's Ringers Solution and
placedwith the mesenchyme
side down on collagen coated Transwell filters above culture
medium (Dulbecco's modified Eagles medium containing 10%
fetal calf serum and antibiotics). The control explants were
treated with 5µl of a solution of 45% 2-
Hydroxypropyl-ß-Cyclodextrin (HPCD) while experimental
explants were treated with 5µg of Cyclopamine in
5µl 45% HPCD. Cultured explants were photographed using
an Olympus SZX9 stereomicroscope and DP-10
camera.
In
situhybridization.
Explants were fixed and probed for gene expression by
incubation with 20µl digoxigenin (DIG)-labeled chick
shhriboprobe overnight
at high stringency essentially as previously described
(Riddle 1993). Regions
ofshhexpression
were visualized with anti-DIG antibody coupled to alkaline
phosphatase followed by bromochloroindole phosphate and
nitroblue tetrazolium in 100 mM Tris pH 9.5, 150 mM NaCl, 25
mM MgCl containing 2 mM
levamisole.
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