• Whole-mount in situ hybridization for the detection of RNA in C. elegans embryos

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B. lacZ fusion RNAs
Because of their great abundance, RNAs derived from chromosomally integrated lacZ fusions are an excellent target for in situ hybridization. When first transcribed, these RNAs accumulate in two nuclear foci, which may correspond to the sites of transcription on the two homologous chromosomes that carry the array (Seydoux and Fire, 1994). The appearance of these "double dots" can help determine the earliest onset of transcription for a gene of interest. Double dots can occasionally also be seen for endogenous RNAs, but are in general more difficult to detect. In contrast to endogenous RNAs that quickly accumulate in the cytoplasm after their initial appearance in the nucleus, lacZ fusion RNAs remain predominantly nuclear and appear quite labile until the 26-cell stage (Seydoux and Fire, 1994). After that stage, lacZ fusion RNAs accumulate in the cytoplasm and become more stable, often perduring longer than endogenous RNAs. This behavior of lacZ fusion RNAs may be due to the long, intron-less coding region of the lacZ gene.

C. Background vs authentic staining.
A common problem associated with the protocol presented here is the high incidence of non specific sticking of the probe to embryos. Often, up to 50% of all wells in an experiment exhibit some form of non-specific staining. This problem may be due to variability in the permeabilization of embryos introduced during the freeze-cracking step. Fortunately, this non-specific staining is easily distinguished from authentic staining. Non-specific staining usually appears within 10 minutes in the color reaction as dark purple patches on the surface of embryos or in nuclei. The best way to distinguish authentic staining from non-specific staining is to compare staining patterns obtained from both antisense and sense probes. Any staining common to both probes is likely to be due to non-specific background. In our experience, successful hybridization with sense probes yields embryos with no staining at all.

VI. Trouble-shooting

In the table below, we list suggestions to limit the occurrence of non-specific staining and other common problems.

Problem	Cause	Solution
Non-specific staining: patchy or uniform staining on the surface of embryos, and/or staining of all nuclei.	Embryo clumping	Limit hypochlorite treatment to minimum amount of time needed for removal of adult and larval carcasses. Before transfering the embryo suspension to the slide, use a micro-pipet to blow air in the suspension to break any clumps.
	Poor freeze-cracking step	Embryos should be in small volume of PBS (10-20µl for a 14x14mm well). Avoid having small pieces of agar on the slide. Use more than one well for each experiment. (4 wells usually guarantees at least one good freeze-crack.)
Non-specific staining: faint staining all over embryos	Excessive probe concentration	Reduce concentration of probe (Concentrations in the range of 0.5-2.5 ng/µl are recommended for most RNAs.)
Non-specific staining: faint staining in extruded portions of embryos	This appears to be an intrinsic property of the Boehringer anti digoxigenin AP-conjugated antibody.	Only intact embryos should be examined.
No signal	Oversquashed embryos	Do not press too hard on the coverslip when freezing the embryos.
	Over or under Proteinase K digestion	Vary concentration and/or duration of proteinase K digestion. Note: Proteinase K digestion can boost the signal obtained from RNAs in lima-bean and older embryos. Proteinase K digestion is not recommended for pre gastrulation embryos.
	Low probe concentration	Increase probe concentration.
No embryos left on slide	Poor freeze-cracking step	Reduce volume of PBS used in freeze-cracking step
	Not enough poly-lysine	Slides are best when subbed with poly-lysine solution the day of the in situs.

Acknowledgments:

We are grateful to Nipam Patel who encouraged us to develop in situ hybridization for C. elegans embryos and generously shared his protocols and expertise. We thank Tom Evans, David Greenstein, Vincent Guacci, Mike Krause, Shohei Mitani, Pete Okkema, Neela Patel and Jorge Mancillas for advice, Susan Strome for her anti-P granule antibodies, David Bird, Dave Hsu, Edward Kipreos, Verena Plunger, Jim Priess, Ann Sluder, Deborah Roussell and Karen Bennett, Patty Wohldmann and Bob Waterston for DNA clones, and Bill Kelly and Peter Okkema for critical reading of the protocol.