Biology Reference
In-Depth Information
is used for microscopy, but longer extrusions may be useful for biochemical studies.
For imaging, spacers are made by cutting 22
22-mm 0 thickness coverslips into
5
22-mm sections with a diamond-tipped pen and positioning them on either side
of the axoplasm secured by a thin coating of Compound 111 silicon grease (DowCorn-
ing). Compound 111 is stable in the seawater and nontoxic. Do not use high-vacuum
silicon grease, which is not designed to seal against aqueous solutions and will be
extracted. A top 22
22-m 0 thickness coverslip is placed to create a sealed chamber.
The top coverslip may be secured with 1:1:1 mix of Vaseline:lanolin:paraffin kept
fluid at 50-60
C, and this creates a microincubation chamber that will maintain
the axoplasm for hours. The ends of the chamber are normally kept open to allow per-
fusion. Chambers prepared in this way have a volume of approximately 25
l, permit-
ting perfusion of axoplasm with buffers at defined ratio of 1:1 to 1:5, minimizing
dilution of axoplasm and allowing introduction of reagents at defined concentrations.
Alternatively, the proximal end may be placed in a small reservoir to extrude into
a buffer of choice (
Morris & Lasek, 1982
). If the extrusion is into a reservoir, the
axon is held in place with the proximal end in the fluid and the rest of the axon
on a clean glass slide. The tubing is slid from the distal end toward the proximal
end. In this case, the axoplasm will extrude into the reservoir while maintaining
the form of the axon (
Morris & Lasek, 1982
).
After extrusion, we routinely incubate the axoplasms in a humidified chamber at
4
C for 15-30 min, which allows the axoplasm to “rest” after extrusion. Empiri-
cally, this produces more reproducible measurements.
Choice of buffers is critical for study of
in situ
microtubule properties. The axo-
plasm is unique in that the small molecular weight components of the cytoplasm can
be directly measured (
Table 9.1
)(
Deffner & Hafter, 1960a, 1960b; Morris & Lasek,
1982
). Standard buffers used for study of microtubules
in vitro
such as BRB80
(80 mM PIPES, 1 mM MgCl
2
, 1 mM EGTA, pH 6.8) and others (
Borisy,
Marcum, Olmsted, Murphy, & Johnson, 1975; Olmsted & Borisy, 1975;
Weisenberg, 1973
) diverge significantly from
in vivo
conditions. Analysis of axo-
plasm shows that
in vivo
conditions have high levels of organic anions (such as
amino acids) and K
þ
is the major cation (
Brady, Lasek, & Allen, 1982; Brady
et al., 1985; Morris & Lasek, 1982
). Axoplasm is a highly reducing environment,
consistent with observations in other cell types. Although total Ca
2
þ
is 3.5 mM, free
Ca
2
þ
levels are much lower, on the order of 100 nM. The axoplasmic cytoskeleton is
sensitive to both ionic composition and ionic strength (
Brady et al., 1985; Brown &
Lasek, 1990, 1993
).
An alternative buffer, buffer X (
Table 9.2
), was developed which retained the
major biochemical features of the axoplasmic milieu (
Brady et al., 1985
). Potassium
aspartate serves as the major organic anion with glycine representing the other amino
acids. Taurine is the major reducing agent and betaine serves as an organic osmolyte.
EGTA is added to buffer the free Ca
2
þ
at 50-100 nM. Halides (F, Cl, Br, and I) may
be problematic in many studies as they can alter protein-protein interactions signif-
icantly (
Collins, 2004, 2006; Hearn, Hodder, & Aguilar, 1988; Westh, Kato,
Nishikawa, & Koga, 2006
) and disrupt the axoplasmic cytoskeleton (
Baumgold,
m
