Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 11

The straight razor blade should be laid down completely and
slide smoothly when drawn along the glass plate. Be careful
not to touch the glass plate with blade edge as it can be easily
damaged this way.
4. Keep the specimen moist all the time. Wet it with small drops
of water from brush to prevent drying and allow sections to
fl oat effortlessly up onto the razor blade (Fig. 1b ).
5. Add drop of water on blade and collect fl oating sections with
fi ne brush (or dropper in case of very small specimens) for fur-
ther processing.

1. Fix samples as indicated above. Label samples with pencil on
slip of cardboard, as graphite lead is stable in any solvent. The
cardboard will pass together with samples through the dehy-
dration and infi ltration series and will be fi nally embedded into
paraffi n.
2. Wash the fi xative out of your samples for 2 × 15 min. For wash
use the same water content as used in the fi xative. In case of
formaldehyde, use the buffer included in the fi xative, for FAA
use ethanol of approximately the same concentration as in the
fi xative.
3. Gradually dehydrate objects and exchange dehydrating solvent
for paraffi n-dissolving intermedium. Thorough dehydration is
indispensable for later successful paraffi n infi ltration. Starting
point of the dehydration series should be selected according to
fi xative as in previous step (e.g., third step of ethanol-butanol
series for 50 % FAA). Pass samples through higher steps of
ethanol-butanol dehydration series with adequate time in each
dehydration step ( see Note 11 ). The sample MUST NOT dry
out during any dehydration step. Use adequate volume of
dehydration solution comparative to sample volume to keep its
dehydration capacity ( see Note 12 ). At least 100× sample vol-
ume might be a good thumb rule.
4. Repeat the anhydrous butanol bath (2× in total) to completely
remove remaining ethanol from samples before starting paraf-
fi n infi ltration.
5. Gradually introduce paraffi n to fully infi ltrate the objects and
exhaustively eliminate butanol (or any other intermedium)
from the samples in the end. Too rash infi ltration is the most
common reason of object shrinkage. That is because butanol
escapes faster from the object than paraffi n is able to replace it
and compensate for volume changes. Timing of individual
steps presented below is informative and should be adjusted
according to the object. Place samples in 100 % (waterless)
butanol in suitable vessels (we use 50 ml vessels with cap for
infi ltration). Add chips of paraffi n (approx. 1/5 of butanol vol-
ume) and let them stand for 1 day at laboratory temperature.

3.5 Paraffi n
Embedding
and Sectioning