Biology Reference
In-Depth Information
Table 21.1
Summary of Procedure, Time Estimates and Points of Interruption
Interruption of Procedure Upon
Completion of Step and Storage
Step
Time
Purification of cells and fixation
in 2% PFA
45 min-4 h
Yes, store in PBS
0.1% PFA at
4 C for up to 3-4 days
þ
Immunogold labeling,
preparation of mica
2h
No
Yes, store overnight in PBS at 4 C
Adherence to mica and
postfixation with 0.1%
glutaraldehyde
2 h
Ultrarapid freezing of samples
1 h
Yes, store indefinitely under liquid
nitrogen
Freeze-etching and replica
generation
3h
No
Floating of replicas on domestic
bleach
30 min
Yes, leave floating overnight at room
temperature
Washing and mounting of
replicas on grids
4 h
Yes, store indefinitely at room
temperature protected from dust
that may have been present in the antibody stock. The ability of the primary antibody
to recognize the antigen after 2% PFA fixation and saturating antibody concentra-
tions should be determined beforehand (e.g., by flow cytometry). Prepare specificity
controls by labeling samples with equal concentrations of isotype-matched irrelevant
antibodies. Wash the cells twice with PBS
þ
0.1%BSA, centrifuging them for 90 s in
a microcentrifuge at 6000 rpm. Samples are then incubated for 1 h with 10 nm gold-
conjugated protein A in PBS
0.1% BSA at previously established saturating con-
ditions (see the succeeding text) and washed twice with PBS þ 0.1% BSA and once
with PBS only. Resuspend cells at a concentration of 4 10 6 cells/ml in PBS.
þ
21.3.3 Cell adherence to freshly exfoliated mica sheets
Directly after starting the first step of the immunogold labeling, mica sheets are cut in
approximately 1 cm
0.5 cm rectangular pieces and split using fine-tipped tweezers.
The split mica pieces are positioned with the freshly exposed surface facing upward
on a piece of parafilm, secured to the bottom of a 10 cm bacterial culture dish. Cut off
the one corner of the mica with scissors (necessary for identification and orientation
purposes during mounting of the mica on the specimen table) and then overlay the
surface with 50
g/ml (w/v) poly- L -lysine solution, prepared in H 2 O.
Incubate for 1-2 h on ice. Wash the mica surface by first removing the poly- L -lysine
solution and then adding and removing three times 50
m
lofa100
m
m
l PBS with a pipette. Be care-
ful to avoid that the solution spills over the sides of the mica. Add directly after the
last wash 50 m l of the immunogold-labeled cell suspension (corresponding to 2 10 5
cells) and incubate for 1 h on ice to allow the cells to adhere to the mica sheet. Gently
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