Biology Reference
In-Depth Information
microscopic control, with a microforge (e.g., as described by
Thomas (1982)
.In
brief, the microscope body is laid on its back so that the stage (removed) would be
vertical and the eyepieces are oriented upwards. A long-working distance objective
(40
) is used and the ME and the ligand vial are held independently by two
micromanipulators.
Depending on glass, shape, and tip diameter 10-30 min of negative pressure is
typically required to fill the tip. Vacuum is slowly released before lifting the
electrode from the ligand. A column height of less than 300
m
m is preferable,
because it decreases the electrode sensitivity to changes in temperature and level
of the bath in the experimental chamber (
Vaughan-Jones and Kaila, 1986
). Our
experience is that, depending on the tip diameter, column heights between 50 and
250
m
m yielded acceptable electrode responses.
Once the electrode is filled with both the ligand and the electrolyte, we prefer not
to let the ME equilibrate in Tyrode or high pCa solutions, because this favors the
deposition of dirt on the tip of the ME and might contribute to clogging. Rather,
we place them, tip up, in a drilled plastic plate, protected by an upside down glass
beaker. Before calibration, the column height is rechecked because THF in the
column continues to evaporate, leading to shrinkage of the PVC gel.
As THF evaporates, the stock solution becomes even thicker. Periodically
enough THF must be added to decrease the viscosity of the mixture. This process
is hastened by mixing it with a glass rod and then on a Vortex mixer (maximal
setting). Sonication can also be used, but does not give better results.
5. Double-Barreled Ca
2
þ
-Selective MEs
For Ca
2
þ
-selective MEs measurements, one must measure both the potential of
the Ca
2
þ
electrode and the local voltage (typically with a KCl-filled ME; see
below). Both electrodes can be built into a single double-barreled electrode,
where one barrel is the Ca
2
þ
electrode and the other is the voltage electrode. We
have not had much luck using these for intracellular recording, but they can be
extremely useful for measurement of local extracellular or interstitial [Ca
2
þ
]in
multicellular preparations (
Bers, 1983, 1985, 1987; Bers and MacLeod, 1986;
Shattock and Bers, 1989
).
Double-barreled electrodes can be pulled from 2 to 2.5 mm diameter theta-style
tubing (R and D Optical Systems, MD) on a Brown-Flaming P-77 micropipette
puller (Sutter Instruments, CA, USA). For extracellular recording, the tips of the
electrodes are carefully broken under microscopic observation to have 4-12
m
m
overall tip diameters. Two methods of silanization of the Ca
2
þ
barrel are practical.
(1) Distilled water is injected into the reference barrel. A hypodermic needle
containing tri-n-butylchlorosilane is introduced into the Ca
2
þ
barrel,
1
m
lof
silane is displaced into the shank of each electrode, and electrodes are placed
in a 200
C oven, tips up, for 5 min and then cooled. (2) a stream of silanizing N,
N-dimethyltrimethylsilylamine (TMSDMA vapor is passed through the Ca
2
þ
barrel (with or without warming), while a stream of nitrogen gas is passed through
