Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces - Plant Cell Morphogenesis: Methods and Protocols - page 101

3.2 Obtaining
the Mold

1. Mix rapidly, for circa 10 s, two small equal-volume drops of
the dental polymer pastes on a lipid-free smooth surface (e.g.,
petri dish cover) using a toothpick. Mix the polymer directly
before usage, preferably when the organ surface is already
exposed. Prepare the mixture of the dental polymer separately
for each mold to ensure that it is fl uid enough during
application.
2. Quickly (as soon as possible), during less than half a minute,
apply a small amount of the polymer on the studied surface
using a toothpick with a sharpened tip or a dental fi lling instru-
ment or excavator. The size of toothpick or the instrument
endings needs to be adjusted to the size of the surface of
interest.
3. After a few minutes check with your fi nger whether the rem-
nant of mixed polymer on the petri dish is no longer sticky. If
it is not, check gently the mixture applied on the organ sur-
face—it may set for a bit longer time than the one on the dish.
As soon as the polymer is not sticky slowly remove the mold
with forceps pulling gently one of its margins from a side so
that the mold is removed gradually, not from the whole sur-
face at once. The latter may lead to organ breakage. Put the
mold in a safe place (no dust or rapid air movement), e.g., in a
petri dish ( see Note 6 ).
4. If you were using a scaffolding, allow the organs, which were
bent away, to move back to their original position, releasing
the threads with forceps.
5. If you are making a sequence of replicas, put a drop of distilled
water on the organ surface from which replica has been taken
or put a transparent cover over the plants to ensure a high
humidity.
6. Attach the mold to a glass slide, putting a drop of a silicon
sealant on a lipid-free glass slide and placing the mold in the
drop. Make sure that the mold is in such position that the
orientation of the surface of interest is more or less horizontal
and that the later-applied resin ( see Subheading 3.3 , step 2 )
will not all fl ow away. Be careful since the sealant sticks really
well to the dental polymer, also to the surface of interest (!).
Because the resin will not set in presence of acetic acid vapor
coming from the sealant, leave the glass slide in an open space
(never in a closed small container) overnight before fi lling
with resin.
7. Repeat steps 1 - 6 to obtain the sequence of molds from the
surface of interest. The number of repetitions and the time
interval between consecutive molds depend on the organ
studied ( see Note 7 ).