Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 19

“jump” and melt onto the slide within few seconds (Fig. 3b-e ).
If you press the slide on section, the section melts on the blade
holder and it is not easy to clean it off.
13. Brush away the condensed ice from blade holder before fur-
ther sectioning.

4 Notes

1. There are two commonly used options of FAA regarding to
fi nal ethanol concentration −70 % and more delicate 50 %
(v/v). Content of acetic acid can be also modifi ed between 2
and 6 %. Material can be stored in solution for considerable
period of time. Use formalin (commercial ~40 % formaldehyde
solution) for preparation.
2. Buffers used with aldehyde fi xatives must not react with them
(e.g., TRIS, EDTA amino groups will react with aldehydes).
Phosphate, HEPES, PIPES-based buffers, or others of Good's
buffers are recommended. Phosphate might precipitate some
divalent cations (Mg 2+ , Ca 2+ ). Osmolarity of the buffer should
be selected according to particular object. For most of plant
samples, we use 25-100 mM buffers. Be aware that 4 % form-
aldehyde itself is a 1.33 M solution.
3. Melting temperature is closely related to hardiness of the paraf-
fi n. It normally stays between 56 and 58 °C, but 54 and 60 °C
mixtures are available too. Composition of the embedding par-
affi n blends differs mainly in content and composition of plas-
ticizers (plastic polymers) improving sectioning properties and
“hardness.” We prefer the use of paraffi n with minimum or no
additives and stabilizers as we have experienced easier infi ltra-
tion and no separation/precipitation of plasticizers at higher
temperatures. Some of the high polymer content mixtures
seem to be rather sensitive to higher temperatures, and it is
better not to exceed 65 °C. Paraffi n without additives can be
cut down to approximately 5 μ m. It is claimed that with addi-

m are accessible. This might be
valid only for soft plant tissues, and we prefer resin embedding
for semi-thin sections. We do not recycle paraffi n with
additives.
4. Adhesives are compounds used to glue sections on the glass
slides. Unlike animal tissues, plant tissues have relatively lower
protein content, and presence of cell wall and vacuole makes
them less adhesive. That is why they fl oat away from the slides
easily during staining or other processing. Selection of the
right adhesive depends on intended use. Glycerol albumen is
the easiest to use, alum gelatin is standard subbing that holds

tives sections down to 2-3 μ