Biology Reference
In-Depth Information
dissecting seeker or similar thin metal rod in the flame of a spirit burner and punch
three holes evenly spread around the side of a 2 ml cryovial, just below the thread for
the screw cap. Also punch three holes through the top of the cap of the cryovial. This
procedure assures that in upright position, the cryovials will keep covering the sam-
ple with liquid nitrogen during transfers and will fill up instantaneously upon sub-
mergence in liquid nitrogen.
21.3.4.3
Freezing of the samples
Once the cryogen is in the plunge freezer, fill a liquid nitrogen bench Dewar to the rim
and put in sufficient cryostorage canes to hold all the samples that are going to be frozen.
Fill an uncapped perforated cryovial with liquid nitrogen, and put the filled cryovial in
upright position in the holding device in the chamber of the freezer unit. Take a sample
containingmica from the 24-well plate using fine tweezers and carefully remove excess
liquid by touching the edge of the mica against a piece of blotting paper for 2-5 s. Im-
mediately, a freshly exfoliated piece ofmica of the same size but with intact corners and
lacking poly-
L
-lysine treatment is placed with its freshly exposed surface against the
cell-containingmica surface. Thismica “sandwich” is held by tweezerswith a clamping
ring. Load the spring of the forceps injector of the plunge-freezing unit and insert and
secure the tweezers with the sample. If the plunge-freezer design allows it, turn the for-
ceps injector away from the freezing chamber when inserting the tweezers with sample.
Position the loaded injector inavertical positionabove the freezingchamber, remove the
cover, and immediately release the sample into the cryogen. The sample will freeze al-
most instantaneously. With the injector still in its down position, remove the tweezers
with sample, blot away excess cryogenwith the piece of filter paper located in the freez-
ing chamber, and transfer the frozenmica sandwich into the liquid nitrogen-filled cryo-
vial, taking care to keep the frozen mica sandwich all the time below the rim of the
freezing chamber. Screw the perforated cap on the cryovial using two pairs of tweezers,
transfer the cryovial to the liquid nitrogen-filled Dewar, and snap the cryovial in a stor-
age cane. The samples can be stored in a liquid nitrogen storage tankuntil themoment of
the cell surface replica generation.
To minimize the risk of ice recrystallization, care must be taken to avoid acciden-
tal warming of the frozen specimens. Manipulation of the frozen samples should be
done using fine tweezers whose tips have been precooled in liquid nitrogen and you
should work fast.
21.3.5
Preparation of cell surface replicas
21.3.5.1
Preparation of the freeze-fracture unit
Precise details of the freeze-fracture unit, evacuation and cooling down, gun cleaning
and adjustments, etc. will vary between different models of freeze-fracture units and
should be conducted according to manufacturer's instructions. However, the proce-
dures and recommendations based on our experience with Balzers BAF 400T
(
Severs, 2007
) and BAF060 machines (
Fig. 21.2
, left) should be of general applicabil-
ity. Carefully clean the electron evaporation beam guns and insert the electron beam
