3. Do the washing and picking-up under the stereomicroscope to
prevent losing embryos. Pick up different corners of the small
paper towel when embryos are washed from one corner to
another.
4. Plastic bristles are hydrophobic like the vitelline membrane so
usually the embryo sticks to the bristle without problems. For
the one-bristle brush, the thicker it is, the easier it is to pick up
embryos as the contact area between the bristle and embryos is
larger. Obviously, bristles cannot be too thick as it becomes
difficult to manipulate. One way to make such a brush is to cut
one bristle off from the root and stick the tip of this bristle back
to the other end of the pen brush. This way the root, which is
thicker, becomes the tip of the one-bristle brush. Flattening the
bristle also increases its area.
5. During this step, once the embryos are on the glass they are
exposed to dry air. It is preferable to do this quickly as possible
to avoid drying the embryos too much. As the orientation of
the embryos will be adjusted later, landing the embryos on its
dorsal side is often the safest and easiest way. Due to its geometry, the dorsal side is physically sturdier and requires less care.
It is possible to place many embryos down on the glass within a
short period of time without damaging them or pushing the
embryos to roll inside the vitelline membrane.
6. It is crucial to orient the embryo precisely so that between the
desired tissues and objective there is only immersion oil, glass,
and vitelline membrane. Again, to decrease the chance of the
embryos rolling inside the vitelline membrane, avoid rolling
the embryos from side to side. Rocking the embryos from
anterior to posterior is more acceptable. If the embryo does
not land precisely in the middle of the ventral side, lift the
embryo up from anterior and before the posterior leaves the
glass adjust the paint brush angle to place the embryo down
from posterior to anterior.
7. Placing an air permeable membrane on the well has several
functions. It maintains the embryos in a very thin layer of
water with sufficient air exchange. Meanwhile it prevents this
thin layer of water from evaporation too fast while imaging.
Furthermore, as this membrane sticks to the well due to atmosphere pressure, there is no worry that the water may spill out
when transported to the microscope and placed on the stage. It
also allows imaging through upright microscope, as long as
there is sufficient working distance, since the petri dish can be
placed upside down with the membrane holding up the water
in the well.
Live Imaging of EMT in Chick Neural Crest
75
prevent losing embryos. Pick up different corners of the small
paper towel when embryos are washed from one corner to
another.
4. Plastic bristles are hydrophobic like the vitelline membrane so
usually the embryo sticks to the bristle without problems. For
the one-bristle brush, the thicker it is, the easier it is to pick up
embryos as the contact area between the bristle and embryos is
larger. Obviously, bristles cannot be too thick as it becomes
difficult to manipulate. One way to make such a brush is to cut
one bristle off from the root and stick the tip of this bristle back
to the other end of the pen brush. This way the root, which is
thicker, becomes the tip of the one-bristle brush. Flattening the
bristle also increases its area.
5. During this step, once the embryos are on the glass they are
exposed to dry air. It is preferable to do this quickly as possible
to avoid drying the embryos too much. As the orientation of
the embryos will be adjusted later, landing the embryos on its
dorsal side is often the safest and easiest way. Due to its geometry, the dorsal side is physically sturdier and requires less care.
It is possible to place many embryos down on the glass within a
short period of time without damaging them or pushing the
embryos to roll inside the vitelline membrane.
6. It is crucial to orient the embryo precisely so that between the
desired tissues and objective there is only immersion oil, glass,
and vitelline membrane. Again, to decrease the chance of the
embryos rolling inside the vitelline membrane, avoid rolling
the embryos from side to side. Rocking the embryos from
anterior to posterior is more acceptable. If the embryo does
not land precisely in the middle of the ventral side, lift the
embryo up from anterior and before the posterior leaves the
glass adjust the paint brush angle to place the embryo down
from posterior to anterior.
7. Placing an air permeable membrane on the well has several
functions. It maintains the embryos in a very thin layer of
water with sufficient air exchange. Meanwhile it prevents this
thin layer of water from evaporation too fast while imaging.
Furthermore, as this membrane sticks to the well due to atmosphere pressure, there is no worry that the water may spill out
when transported to the microscope and placed on the stage. It
also allows imaging through upright microscope, as long as
there is sufficient working distance, since the petri dish can be
placed upside down with the membrane holding up the water
in the well.
Live Imaging of EMT in Chick Neural Crest
75