Biology Reference
In-Depth Information
3.6 Cryosectioning
1. Fix the specimen in an appropriate fi xative (e.g., 4 % formalde-
hyde in phosphate buffer). Use lowered pressure (“vacuum
infi ltration”) to substitute fi xative for the air within the tissue if
necessary.
2. Wash out fi xative for 15 min with phosphate buffer used to
prepare fi xative.
3. Infi ltrate samples gradually with 3, 10, and 20 % sucrose solu-
tions. Each step takes at least 30 min at room temperature.
Agitate gently and apply 0.1 % of surfactant (Triton or Tween
20) with 3 % sucrose solution to facilitate infi ltration. Individual
steps should be prolonged to infi ltrate properly compact and
more voluminous samples ( see Note 15 ).
4. Freeze pretreated samples directly on the specimen chuck. Let
the cryostat cool down to working temperature and turn on
cryobar (freezing shelf) boost to minimize the bar temperature
fi rst. Add small amount of semisolid cryoembedding medium
(e.g., OCT or high-viscosity cryoembedding medium) on the
specimen chuck and use heat extractor to make fl at base (the
extractor frequently stick to the medium if not properly fro-
zen; apply Tefl on coating spray to the extractor to minimize
this problem). Add more medium on the top of frozen plat-
form and transfer the sample into this medium. Quickly arrange
sample into desired position and freeze the block on cryobar.
Sample should be covered with tiny layer of cryoembedding
medium ( see Note 16 ). Frozen samples can be stored at −80 °C
in closed container if necessary. Do not store them in the cryo-
tome chamber as the samples dry out rather quickly.
5. Trim frozen medium encasing object to adjust the specimen
block size for easier cutting. Leave enough medium in sur-
rounding of the sample. However, it is more diffi cult to cut
thin sections from larger block.
6. Prepare all needed equipment (brushes, forceps, etc.) into cryo-
tome chamber to get to right working temperature before use.
7. Mount the chuck with object on the microtome head and let
its temperature to equilibrate. Working temperature should be
selected according to desired thickness, character of the sam-
ple, and composition of embedding medium beside others.
Independent setup of chamber (knife) and sample temperature
is of advantage; it might be convenient to use 2-3 °C lower
temperature of knife than sample (e.g., [ 43 ]). Commonly we
use specimen temperature between −8 and −20 °C for stan-
dard section of 8-20
m. Thinner sections might require lower
temperature. It is reasonable to start with −15 °C and adjust
the temperature according to appearance of sections. If the
sections wrinkle and smear on knife, the working temperature
is too high. If sections crumble, temperature is too low. For
troubleshooting of the most common problems ( see Table 4 ).
μ
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