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III. Other Methods for Examining the Development of Neurons
Rationale: Although transgenic fluorescent protein labeling is now universally
used to study neuronal development, other methods remain extremely valuable for
revealing particular neuronal features and for validating the effects on endogenous
proteins and native cells.
A. Dye-Filling
C. elegans head amphid and tail phasmid sensory neurons can be labeled by
lipophilic fluorescent dyes, such as fluorescein isothiocyanate (FITC), 1,1-diocta-
decyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), 3,3
0
-dilinoleyloxacar-
bocyanine perchlorate (DiO), and 1,1
0
-dioctadecyl-3,3,3
0
,3
0
-tetramethylindodicar-
bocyanine perchlorate (DiD). The mechanism of dye uptake seems to involve the
exposed endings of sensory cilia and correlates with some aspects of neuronal
function. FITC can be used to label the ADF, ASH, ASI, ASJ, ASK, and ADL cells
of the amphid sensilla, and the PHA and PHB neurons of the phasmid sensillum. DiI,
DiO, and DiD can be used to visualize the ASI, ADL, ASK, AWB, ASH, and ASJ
amphid neurons, as well as the PHA and PHB phasmid neurons. Detailed protocols
for dye-filling experiments can be found in Shai Shaham
'
s review (''Methods in Cell
Biology'') of the wormbook (
http://www.wormbook.org/chapters/www_intro-
B. Immunocytochemistry
Immunostaining using antibodies against various proteins remains invaluable for
analyzing subcellular structures and protein localization in vivo. Numerous antibo-
dies of neuronal proteins such as UNC-17/ChAT, synaptotagmin, and UNC-10/Rim,
mechanosensory microtubules, or neurotransmitters such as serotonin and GABA
produce highly specific and consistent patterns (
Table III
). Avery useful monoclonal
antibody toolkit for nervous system analysis was made by the Nonet lab (
Table III
)
(
Hadwiger et al., 2010
). The major hurdles in C. elegans antibody staining are
antigen fixation and permeating the eggshell of embryos and cuticle of larvae and
adults. The best staining comes from practice and trying out a number of fixation
procedures. Three commonly used procedures are paraformaldehyde-fixation by
Finney and Ruvkun (1990)
, Bouin fixation by Mike Nonet (
Nonet et al., 1997
),
and freeze-crack by Janet Duerr (
Duerr et al., 1999
). For detailed protocols on
antibody staining, see the wormbook chapter ''Immunohistochemistry'' by Janet
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