Electrophysiological Assessment of Cerebral Vasospasm - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 478

Pipette puller and microforge . To precisely control the size and
shape of glass micropipette used for patch-clamping, a multiple
step programmable horizontal puller (such as the Flaming/Brown
micropipette puller P-97, Sutter Instrument) is helpful and has some
advantages over the simple two stage vertical pullers. To achieve a
better seal of the pipette tip over the cell membrane, a microforge
is needed to polish and smooth the tip of the pipettes.
Vibratome . In studies using acutely isolated brain slices, a vibratome
is needed to produce viable slices. There are two major vibratome
makers, namely Leica Microsystems (Wetzlar, Germany) and The
Vibratome Company (Bannockburn, IL).

Grounding electrode . This is usually made from a silver wire and
connected to the grounding outlet of the headstage through a pin.
The silver wire should be chloride-coated by simply using household
bleach. Alternatively, the silver wire can be connected to an agar
bridge, which is made from a U-shaped capillary tube fi lled with
agar that is made with the bath solution.
Glass capillaries . Capillaries are used to make the electrodes for
patching on cells. Different types of glass can be used. The key is to
choose glass that produces the least noise or at least less than that
of the electrode. In this regard, borosilicate and aluminosilicate
glass are the most commonly used. Quartz glass has the lowest
noise, but it is more expensive and requires a special laser puller to
produce the electrodes. It can be used for extreme low noise
recording, such as single channel recordings.
Buffer solutions . In general, the bath solution for isolated cells is
composed of a solution designed to mimic the extracellular solu-
tion in vivo, which is typically high in sodium (Na + ). Conversely,
the solution used to fi ll the patch pipette (recording electrode) is
composed to mimic the intracellular contents, which has a high
concentration of K + . For example, the bath solution used in our
laboratory contains (in mM): NaCl 100, KCl 50, MgCl 2 1, CaCl 2 2,
HEPES 10, and glucose 10, pH 7.4. The internal (patch pipette)
solution consists of (in mM): KCl 142.6, KOH 2.4, MgCl 2 2.5,
BAPTA 10, and HEPES 10, pH 7.2 ( 7 ). In addition, these solutions
could be modifi ed according to experimental purposes depending
on what ionic currents one is trying to record. For example, BAPTA
was included in the pipette solution to buffer intracellular Ca 2+ to
completely block large conductance, Ca 2+ -activated K + channel
currents in the study of inward rectifying K + currents ( 7 ). There are
numerous other variations in terms of solutions and also of the
confi guration of the patch-clamp system. In some studies, 200 mg/ml
of nystatin was added to perform nystatin-perforated patch tech-
nique to measure the resting membrane potential in the current-
clamp mode ( I = 0) ( 7 ). Drugs to block some channels may be used
in order to isolate currents due to other channels ( 1 ).

2.2. Materials Used
for Patch-Clamping