Biology Reference
In-Depth Information
view. There are two principal approaches to minimize freezing
distortion—fl ash freezing or cryoprotection pretreatment. Flash
freezing approach prevents formation of large hexagonal ice crys-
tals due to high freezing rate and small cubic crystals or even vitre-
ous ice should form. Effi ciency of the procedure can be further
increased under high-pressure conditions [ 32 ]. Isopentane super-
cooled with liquid nitrogen or with solid carbon dioxide is fre-
quently used to ensure proper heat transfer from object.
Supercooling is suitable only for small specimens and even in such
a case low thermal conductivity of biological samples presents
the limitation for freezing rate [ 33 ]. The other approach restricts
formation of large ice crystal due to presence of rather high con-
centrations of cryoprotective solutes. Sucrose is common cryo-
protectant used in wide range of concentrations from 10 to over
75 % [ 34 - 36 ]. Infi ltration with 8-15 % glycerol [ 37 ], 10 %
dimethyl sulfoxide [ 38 ] or polyvinyl alcohol, and polyethylene
glycol mixtures [ 39 ] can be used. Freezing rate of the cryopro-
tected specimen is far less critical, and freezing directly in the
cryostat chamber (freezing shelf) is thus possible. The process of
antifreeze treatment takes several hours and therefore requires
foregoing fi xation of samples to minimize processing-related arti-
facts. Besides freezing of the object, proper setup of cryotome
(temperature, anti-roll plate, blade settings) is crucial for success-
ful sectioning.
2
Materials
2.1 Fixation
1. FAA (formalin-acetic acid-alcohol): Mix together 50 % (v/v)
of ethanol, 5 % (v/v) of acetic acid, 5 % (v/v) of formalin, and
40 % (v/v) of distilled water (should be adjusted according to
stock ethanol and acetic acid concentration; for variations see
Note 1 ).
2. 4 % formaldehyde in 50 mM phosphate buffer (pH 7.2):
Dissolve 8 % (w/v) of paraformaldehyde (PFA) in distilled
water; to facilitate dissolution, add minimal volume of 1 M
KOH solution (approx. 200
l per 100 ml) and warm the solu-
tion up to ~60 °C in a fume hood. When PFA is dissolved (the
solution comes clear), add equal volume of 100 mM phos-
phate buffer of proper pH (selected according to purpose).
Check pH and titrate to required pH with 1 M HCl if neces-
sary ( see Note 2 ).
3. Phosphate buffer: Mix together x ml of 0.2 M acid sodium
phosphate (27.8 g NaH 2 PO 4 in 1,000 ml) + y ml of 0.2 M
middle sodium phosphate (53.65 g Na 2 HP0 4 .7H 2 0 in
1,000 ml) fi ll up to 200 ml with distilled water to gain 100 mM
buffer. The values of x and y are specifi ed in Table 1 .
μ
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