Biology Reference
In-Depth Information
B. Hypaque meglumine
Bacterial contamination (usually E. coli OP50 from plates) often forms clumps
around worms and deposits onto slides, attracting probe and contributing to high
background. In our hands, cleaning worms with meglumine gives the most consis-
tent results. It may be possible to bypass this step if the source plates are clean
enough (David Fitch, personal communication). Alternatively, sucrose flotation may
be tried (suspend animals in 30% sucrose at 4
C and centrifuge at 4000 rpm).
C. Nontoxic Fixative
Although formaldehyde is a widely used fixative, NTF is an alternative to form-
aldehyde that is easily made, stable, and nontoxic. More importantly, we have found
it to greatly improve sensitivity and preservation of fine structure, and it appears to
be less prone to overfixation. This protocol was originally developed using STF
(Streck Tissue Fixative; Streck Laboratories;
Montgomery et al., 1998
), but this
reagent has been discontinued. NTF is based on the composition of STF (documen-
ted in United States Patent 5460797) and in our hands appears to be equally effective.
D. Purchase of Ready-Made Reagents
Many of the reagents in this protocol, such as 2
SSC and 1 M Tris-HCl, can be
easily made. The ready-made forms are purchased only to make it less likely that
RNase contamination might be introduced.
E. Labeling of Nuclei
We have found that use of fluorescence (e.g., to visualize DAPI) causes the purple
color to develop very rapidly in a nonspecific manner. If the slides are dehydrated
and mounted in a permanent mounting medium (e.g., Permount with DAPI) the
nuclei can be visualized, although the morphology is adversely affected.
F. Staining Small Quantities of Embryos or Other Stages
A larger number of animals is necessary for consistent freeze-cracking. When the
number of specimens is limiting, sterile adults of recognizable body morphology
could be added (e.g., Dpy; Glp). If it is desired to stain larvae or adults, they should
be synchronized so that they are all the same size prior to mounting on the slides.
G. Staining of Other Species
We and others have been successful in staining other nematode species, includ-
ing C. remanei
and C. briggsae
(
Coroian et al., 2005; Lin et al., 2009
)and
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