Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 20

well the sections, and Poly l-lysine is good solution for immuno
and other more sensitive applications. To prepare adhesive
(subbing)-coated slides, cleaning and degreasing of slides is of
high importance. Even new slides should be washed with
detergent followed with 96 % ethanol and distilled water.
Alternatively the slides can be washed in dishwasher and care-
fully rinsed with distilled water before use.
5. Cryoembedding media are commercially available or can be
prepared in the laboratory. We have positive experience with
both options. OTC (optimum cutting temperature com-
pound) is a commercially available cryoembedding medium
(e.g., Tissue-Tek OCT) based on polyvinyl alcohol (PVA) and
polyethylene glycol (PEG). Cryo-gel TM , Cryomatrix TM , and
PolyFreeze TM are further commercial options differing in
viscosity.
6. It might be accepted as a common rule of thumb that volume
of fi xative should not be less than 50× volume of the fi xed tis-
sue. Otherwise, the buffering capacity of the solution (pH,
concentration of fi xative, molarity) might not be suffi cient.
7. There are very few experimental data to estimate the time
needed for aldehyde penetration and fi xation. In animal tissues
(e.g., liver), the penetration rate normally does not exceed
1 mm per h. In the work of Mersey and McCully [ 8 ], the acro-
lein fi xation passed about 140 m per min along the root hair.

The formation of linkages (incorporation of formaldehyde)
within the tissue might be also rather slow process, taking
hours to be saturated (e.g., [ 5 ]).
8. Rotary vane vacuum pump with pressure regulator and plastic
desiccator allow for controlled gradual drop of pressure within
the desiccator chamber. Evaporating fi xative (or any other
solution) accumulates within the oil and can cause corrosion
(damage) of the pump chamber. It is necessary to let the pump
run long enough to warm up the oil and evaporate the con-
densate from pump. It is condensate induced corrosion
induced corrosion that most frequently damages the rotary
vane vacuum pump. During vacuum infi ltration, make sure to
vent pump exhaust into the fume hood and not into labora-
tory. Fixatives are toxic. If the fi xative fi xes your samples, your
own tissues might be fi xed as well!
9. Tissues of high density or pigmentation might require remov-
ing the cytoplasm content with 2 % NaOH in 30 % EtOH or
dimethyl sulfoxide. The latter is more effi cient and can remove
complete protoplasts. Extraction of chlorophyll and lipidic
compounds might be done in methanol/chloroform (1:1)
mixture. To clear colored phenolic depositions, alkalized
hydrogen peroxide or sodium hypochloride-based protocols
mentioned in introduction should be used.

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