Biology Reference
In-Depth Information
remove the solution from the top of the mica by aspirating with a pipette positioned in
one of the corners of the mica. Wash two times with 50
l PBS, adding the PBS with
a pipette positioned in one corner and aspirating with the pipette positioned in the
diagonally opposing corner. Perform a postfixation by adding 50
m
l of a freshly pre-
pared solution of 0.1% (v/v) glutaraldehyde in PBS. Incubate for 20 min on ice and
wash three times with 50
m
l PBS, using the “diagonal” washing procedure described
earlier. Store the samples at 4
C in individual wells of a 24-well tissue culture plate
filled with PBS until the freezing step.
m
21.3.4
Freezing of samples
21.3.4.1
Preparation of the plunge-freezing unit
We use a Reichert-Jung (now Leica) KF-80 plunge-freezing unit (
Fig. 21.1
) and liq-
uid ethane as secondary cryogen. Alternatively, liquid propane can be used as sec-
ondary cryogen, but one has to take extra precautions as it is much more inflammable
than ethane. Homemade plunge freezers or a Leica CPC unit can be used as well. For
immersion fixation of the mica sandwiches, a forceps injector is used.
This ultrarapid freezing (cooling rates in excess of 10
4
C/s) ensures good ultra-
structure preservation deep as 10-20
m, enough for the freezing of mica sandwich
mounted cells, allowing us to work in the absence of cryoprotectants (
Severs, 1995;
Severs et al., 1995
) such as glycerol, which can induce membrane artifacts and dif-
ficult etching due to its low volatility at low temperatures and under vacuum. Ultra-
rapid freezing can be achieved by plunge freezing or by metal block-impact freezing,
while manual immersion of specimens into the cryogenic liquid does not ensure good
preservation of samples that have not been cryoprotected.
Connect the freezing unit to a liquid nitrogen tank and a gas cylinder containing
the secondary cryogen according to the instructions provided in the manual of the
unit. Make sure that the cryogen container is inserted in its dedicated slot and place
a piece of filter paper and a metal bottle cap or any other metal container that permits
upright positioning of the cryovial in the freezing chamber (
Fig. 21.1
). Cover the
chamber with its lid. Note that the chamber should be covered with the lid between
each cycle of sample freezing. Bring the freezing unit to its desired operating tem-
perature. The freezing point of ethane is
m
187
C. Select a
temp just (1-2
C) above the freezing point of the cryogen to be used. When the op-
erating temperature has been reached, fill the cryogen container with liquid cryogen
up to the top. In plunge freezers lacking temperature control, the cryogen will freeze
at the temperature of liquid nitrogen. One can defrost the cryogen by inserting a
metal object into the cryogen just before plunging the sample.
171
C and of propane
21.3.4.2
Preparation of cryovials for storage of the frozen
mica sandwiches
During the cooling of the plunge-freezing unit, prepare the cryovials to be used for
storage of the samples. Each vial can hold up to two frozen mica sandwiches, pro-
vided that they are experimental duplicates. Label the vials. Heat the tip of a
