Biology Reference
In-Depth Information
b. Methods
ACSF should be prepared fresh on the day of experiments. Two 50 ml falcon tubes
filledwithACSF (Ca
2
þ
free) can be placed at
20
C for approximately 1-2 h prior to
the preparation of brain slices. This partly frozenmedium is used to fill the bath on the
Vibratome where the brain will be sliced. Once the Vibratome is ready, rapidly
remove the brains from neonates, dissect the cerebellum away, and place the brain
ventrally against the agar block. Horizontal or coronal slices (400
m
m) can be cut and
transferred immediately to a small Petri dish containing oxygenated ACSF and
coelenterazine (10
m
M) and incubated at room temp, in the dark for 45-60 mins.
Once the slice has been inserted into the imaging chamber, a slice anchor (Harvard
Apparatus) can be used to secure the tissue. Slices should be continuously perfused (at
a flow rate of 1 ml/min) with oxygenated ACSF containing 2 mM CaCl
2
. Biolumi-
nescence signals can be monitored as previously described (
Rogers et al., 2007
).
C. Summary of Section II
The commercial development of products, kits, or services enabling genetic
engineering of cells or animals means that these technologies are no longer out
of reach to biologists who have experience in imaging but who have little molecular
biology experience. BRET-based imaging depends on the degree of spectral over-
lap, relative orientation, and the distance between donor and acceptor dipoles.
Fluorescent proteins derived from coelenterates or their variants are therefore
likely to be the most suitable acceptors owing to their structural similarity with
GFP (
Tsien, 1998
). The recent development of RFP-aequorin (
Manjarr
´
s et al.,
2008
) has provided the means to simultaneously monitor Ca
2
þ
signals from two
di
V
erent microdomains within a single cell.
III. Introducing Coelenterazines into Cells, Tissues and Embryos
400 Da) prosthetic group that binds with apoae-
quorin to form the active aequorin complex. As coelenterazine is subject to oxida-
tion, it is normally supplied in a sealed vial free of O
2
. Prior to reconstitution, the
coelenterazine should be stored at
Coelenterazine is the small (
20
C. In addition, coelenterazine is poorly
soluble in water and so stock solutions are normally prepared in methanol. In this
form, coelenterazine is stable for
20
C. Coelenterazine was
originally isolated from Aequorea victoria; however, in the 1970s a procedure for
chemically synthesizing coelenterazine was developed (
Inoue et al., 1975; Kishi
et al., 1972
). This procedure has since been used for preparing coelenterazine and
its analogs (
Jones et al.,1999
). Many of the coelenterazine analogs possess proper-
ties di
3 months at
erent from those of native coelenterazine. These include half-life, aequorin
regeneration rate, luminescence capacity, emission maximum and membrane
permeability, the latter being due to the lipophilic nature of coelenterazine.
For example,
V
f-coelenterazine has the same half-life as
native
coelenterazine
