Biology Reference
In-Depth Information
Maddox and Maddox). Here we provide additional details regarding this key tech-
nique as it relates to analyzing morphogenesis. Agar mounts have several key
advantages for analyzing morphogenesis. First, the mount slightly compresses the
embryo, holding it in place. Second, such compression produces a consistent orien-
tation convenient for imaging many aspects of embryonic morphogenesis. As the
processes of morphogenesis proceed, either the dorsal or ventral surface of the
embryowill be against the coverslip. After ventral enclosure is complete, the embryo
then turns on its side, such that every embryo will be positioned with either its right
or left side facing the coverslip. For many morphogenetic events, especially those
involving the embryonic epidermis, such mounts are very useful (see
Chisholm and
Hardin, 2005
for details of the basic movements associated with epidermal morpho-
genesis). For some events, other orientations of the embryo may be preferable, and
for these purposes, other mounting techniques may be used (see below).
Imaging setup
For assembling the mount, a standard stereomicroscope is required. To identify
early embryos (1-4 cell), a total zoom of 80
or greater is recommended. We have
typically used either a Wild MZ5 microscope with 20
oculars or Leica MZ12.5
microscope with 16
oculars.
Materials
i.
Reagents:
Agar (5% w/v)
M9 buffer:
3gKH
2
PO
4
6gNa
2
HPO
4
5 g NaCl
1 mL 1 M MgSO
4
1LH
2
O
Valap:
Equal parts by volume of vaseline, lanolin, and paraffin.
Heat thoroughly until melted and mix.
ii.
Equipment:
Calibrated glass pipettes (50
m
L)
Coverslips (18
18 mm, No. 1)
Eyelash brush (eyelash glued to end of round toothpick)
Mouth pipette 15
00
aspirator tube assembly
Microscope slides (25
75
1 mm)
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