5. Remove the fibronectin solution using a P1000 micropipette
and rinse once with PBS1Â (see Note 5).
6. Rinse the dish with a small amount of DFA1Â to remove traces
of PBS1Â.
7. Fill the dish with DFA1Â.
3.1.2 Microdissection
1. Place embryos at stage 15/16 in a petri dish filled with NAM
0.25Â and remove the vitelline membrane using fine forceps.
Let them recover as superficial wounding may have occurred
and let embryos further develop to stage 18 (see Note 6,
Movie 1).
2. Select embryos at stage 18 (Movie 2).
3. Fill the dish containing the modelling clay with NAM 0.25Â.
Using the glass ball, form small holes in the modelling clay. The
depth of each hole should be roughly half the width of an
embryo (see Note 7, Movie 3).
4. Place one embryo per hole. Turn each embryo slightly on one
side so that the neural crest region to be removed is facing up
(Fig. 2a, Movie 3).
5. Using the glass ball, move gently the clay toward the embryo
until it touches the epidermis making sure the orientation is
preserved (Fig. 2b) then apply pressure all around the embryo
by pushing the clay onto its sides to firmly secure the embryo
(Fig. 2c, Movie 3, see Note 8).
6. Using the hair knife make a small incision posteriorly and
dorsally to the neural crest area (Fig. 2c, step 1, Movie 3).
7. Insert the tip of the hair knife in that initial hole, keeping the
hair parallel to the tissue. The hair knife should be in between
the pigmented layer and the neural tissue (Fig. 2c, step
2, Movie 3).
8. Gently remove the pigmented layer from medial to lateral to
uncover the neural crest region (Fig. 2c, step 3, Movie 3).
9. Place the hair knife parallel to the ventral side of the neural crest
region (dotted line on Fig. 2d). Apply a gentle pressure (do not
attempt to cut the tissue) and move from lateral to medial
repeatedly. The neural crest region will detach from its surroundings (Fig. 2e, e
0 ). Continue until the whole region
detaches by itself (Movie 2). After dissection there should be
a hole in which the underlying mesoderm can be seen (Fig. 2f).
When a dissected embryo is released from the clay the actual
size of the wound should be relatively small and the neural tube
on the dissected side should be intact (Fig. 2g, g
0 ). Look out for
contamination from other cell types (Fig. 3, see Note 9). An
example of a rough dissection including mesoderm is shown at
the end of Movie 3.
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and rinse once with PBS1Â (see Note 5).
6. Rinse the dish with a small amount of DFA1Â to remove traces
of PBS1Â.
7. Fill the dish with DFA1Â.
3.1.2 Microdissection
1. Place embryos at stage 15/16 in a petri dish filled with NAM
0.25Â and remove the vitelline membrane using fine forceps.
Let them recover as superficial wounding may have occurred
and let embryos further develop to stage 18 (see Note 6,
Movie 1).
2. Select embryos at stage 18 (Movie 2).
3. Fill the dish containing the modelling clay with NAM 0.25Â.
Using the glass ball, form small holes in the modelling clay. The
depth of each hole should be roughly half the width of an
embryo (see Note 7, Movie 3).
4. Place one embryo per hole. Turn each embryo slightly on one
side so that the neural crest region to be removed is facing up
(Fig. 2a, Movie 3).
5. Using the glass ball, move gently the clay toward the embryo
until it touches the epidermis making sure the orientation is
preserved (Fig. 2b) then apply pressure all around the embryo
by pushing the clay onto its sides to firmly secure the embryo
(Fig. 2c, Movie 3, see Note 8).
6. Using the hair knife make a small incision posteriorly and
dorsally to the neural crest area (Fig. 2c, step 1, Movie 3).
7. Insert the tip of the hair knife in that initial hole, keeping the
hair parallel to the tissue. The hair knife should be in between
the pigmented layer and the neural tissue (Fig. 2c, step
2, Movie 3).
8. Gently remove the pigmented layer from medial to lateral to
uncover the neural crest region (Fig. 2c, step 3, Movie 3).
9. Place the hair knife parallel to the ventral side of the neural crest
region (dotted line on Fig. 2d). Apply a gentle pressure (do not
attempt to cut the tissue) and move from lateral to medial
repeatedly. The neural crest region will detach from its surroundings (Fig. 2e, e
0 ). Continue until the whole region
detaches by itself (Movie 2). After dissection there should be
a hole in which the underlying mesoderm can be seen (Fig. 2f).
When a dissected embryo is released from the clay the actual
size of the wound should be relatively small and the neural tube
on the dissected side should be intact (Fig. 2g, g
0 ). Look out for
contamination from other cell types (Fig. 3, see Note 9). An
example of a rough dissection including mesoderm is shown at
the end of Movie 3.
262
Nade ` ge Gouignard et al.