Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 12

6. Place caped dishes at 40 °C oven and let paraffi n dissolve. Add
enough paraffi n to keep a few undissolved chips on the bottom
and let over day.
7. Open the dishes in the end of the day and let overnight. If the
paraffi n is completely dissolved add more. Part of the butanol
will evaporate slowly—be careful not to let the samples dry.
8. Increase temperature to 58 °C, add paraffi n, and let stand for
1 day (samples can stand even over weekend in this or latter
steps).
9. Pour off approx. one third of the paraffi n-butanol mixture and
bring to original volume with melted paraffi n. Let samples
infi ltrate for 3 h to half a day.
10. Replace half of the mixture with melted paraffi n (3-12 h).
Repeat this step once more.
11. Let open vessels stand overnight in oven to completely evapo-
rate residuum of butanol. It is convenient to apply low pres-
sure in vacuum oven (paraffi n must not solidify) to facilitate
complete butanol removal. Butanol should not be smelled
from the samples at the end of this step.
12. Replace melted paraffi n for a pure one and let stand for half a
day (paraffi n can be recycled for this step) to eliminate rest of
butanol from samples.
13. Replace melted paraffi n for a pure one and proceed to
embedding.
14. Pour the last paraffi n change with objects into the paper ori-
gami dish (Fig. 2a ) or suitable mold ( see Note 13 ). Top up
with pure melted paraffi n (do not exceed 65 °C) and arrange
the samples on the bottom so that it is easy to separate them
latter (Fig. 2b ).
15. Individual samples or their groups (e.g., sets of root segments)
might be organized into rows and columns. Arrangement can
be achieved with hot needle or tweezers, which are also conve-
nient to remove potential air bubbles from hot paraffi n surface.
Arrangement can be done on hot plate to extend period of
time for manipulation, but fast work in ceramic dishes is usu-
ally suffi cient. Suitable orientation of objects for sectioning has
to be considered during embedding. Cardboard tag should be
placed into the block together with samples.
16. Cool paraffi n blocks. Rapid cooling rate is recommended (e.g.,
cool water bath). Paraffi n blocks can be stored for long time
(many years at room temperature).
17. Cut individual samples from the paraffi n block and fi x them to
wooden, metal, or plastic chucks suitable for your microtome
clamp. Heat the block and chuck at the site of contact to melt
surface layer of paraffi n and press them together.