Biology Reference
In-Depth Information
choice in our lab. Combination of gradual dehydration and
concurrent introduction of intermedia minimizes distortion and
saves time. Damage and/or hardening attributed to longer action
of higher temperature during infi ltration with paraffi n reported in
literature was not observed to be that signifi cant in our hands, but
damage caused by overly fast infi ltration progress or improper
elimination of intermedium was recorded rather frequently.
Protocol of paraffi n oil-regulated rate of infi ltration should be
mentioned [ 29 ] in this context. This protocol use mixture of buta-
nol and paraffi n oil (1:1) instead of pure butanol to saturate the
objects before paraffi n infi ltration. Paraffi n oil has higher viscosity
and is therefore slowly replaced with melted paraffi n in latter steps,
reducing thus shrinkage of tissue related to paraffi n infi ltration.
The whole procedure of sample processing towards paraffi n
embedding is a sequence of events. Each of them might introduce
artifacts into the preparation, which cannot be corrected latter on
and accumulation of errors therefore commonly takes place.
Alternatives to paraffi n sections might be found in low tem-
perature melting Steedman's wax ( see ref. [ 31 ]), which is suitable
for higher temperature sensitive objects (e.g., sections for immu-
nodetection) and infi ltration protocol is signifi cantly shorter. On
the other hand, sectioning, fl attening of sections on slides, and
storage of samples are slightly more complicated with hygroscopic
nature of the polyethylene glycol distearate-based wax. There are
various types of resins used for sample embedding and sectioning
(Technovit, LR White, Lowicryl, GMA, Spurr, etc.) that differ in
hydrophobicity, hardness, and sectioning properties. Resin-
embedded objects can be sectioned to thinner slices (less than
1
m) to achieve higher degree of cytological details. Because of
specifi c requirement for sectioning of the resin, this topic is out of
scope of this chapter.
μ
Sectioning of frozen material does not require extensive sample
dehydration and embedding medium infi ltration. Cryosections are
suitable for a wide range of light-microscopy applications (e.g.,
immunohistochemistry, in situ hybridization, enzyme histochemis-
try), but it should be mentioned that it might not be straightfor-
ward to gain good quality sections for plant tissues. Fixed or fresh
(unfi xed) samples might be processed according to intended appli-
cation. Standard thickness of sections is 8-20
1.5 Sectioning of
Frozen Material
μ
m, but thickness
down to 3
m is attainable for some samples using standard cryo-
microtome. Objects are encased into cryoembedding medium,
which acts as an object-surrounding matrix for sectioning.
Freezing is critical step of the procedure, which strongly
determines quality of sections. Freezing procedure should prevent
formation of large ice crystals inside the sample, structural dam-
age, and related sectioning problems. Highly vacuolated plant tis-
sues are therefore rather complicated objects from this point of
μ
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