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

Whole-mount in situ hybridization for the detection of RNA in C. elegans embryos
Geraldine Seydoux and Andrew Fire

Adapted from Seydoux, G. and Fire, A. (1995). Whole-mount in situ hybridization for the detection of RNA in C. elegans embryos. In C. elegans: Modern Biological Analysis of an Organism. Methods in Cell Biology (ed. H. Epstein and D. Shakes) Academic Press, San Diego.

I. Materials
A. Reagents
- Anti-digoxigenin Fab fragment, rhodamine labeled (Boehringer Mannheim Cat# 1207 750)-
Anti-digoxigenin Fab fragments, alkaline-phosphatase labeled. (Boehringer Mannheim Cat# 1093 274)-
BSA (Fraction V, Sigma Cat# A-7906)-
Commercial Bleach (5.25% Sodium hypochlorite solution, Clorox)-
DAPI (Sigma Cat# D-1388)-
DNA from salmon testes (Sigma, Cat# D-1626) -
Formaldehyde (37%, Fisher, Cat# F79-500)-
Formamide (Boehringer Mannheim Cat# 100 731)-
Glycerol (Boehringer Mannheim Cat#100 649)-
Glycine (Sigma Cat# G-4392)-
Glycogen (Boeringher Mannheim Cat# 901-393)-
Heparin (Sigma, Cat# H-3393)-
Hepes (Boehringer Mannheim Cat# 242 608)-
Levamisole (Sigma, Cat# L-9756) -
NaN3 (Sigma Cat# S-2002)-
NBT: 4-Nitro blue tetrazolium chloride. (Boehringer Mannheim Cat# 1383 213)-
p-Phenylenediamine (Sigma Cat# P-6001)-
0.1% Poly-L-lysine solution (Sigma Cat# P-8920). -
Polyoxyethylene-Sorbitan Monolaurate (Tween 20; Sigma Cat# P-1379)-
Proteinase K (Boehringer Mannheim, Cat# 161 519)-
Taq DNA polymerase (Promega Cat# M1861)-
Tris (Boehringer Mannheim Cat# 604 205, 812 854)-
Triton X-100 (Sigma Cat# X-100) -
X-phosphate: 5-Bromo-4-chloro-3-indolyl-phosphate. (Boehringer Mannheim Cat# 1383 221)

B. Stock solutions
DEPC treatment of solutions is not needed. Solutions are stored at -20oC unless otherwise indicated.-
10x dNTP mix: Digoxigenin-11-dUTP pre-mixed with other nucleotides (1 mmol/l dATP, 1 mmol/l dCTP, 1 mmol/l dGTP, 0.65 mmol/l dTTP, 0.35 mmol/l DIG-dUTP). (Boehringer Cat# 1277 065.) -
10x PBS: 80g NaCl, 2g KCl, 6.1g anhydrous Na2HPO4, 2g KH2PO4, H2O to 1 liter. Autoclave and store at room temperature.-
10x Taq Buffer: 500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25oC), 1% Triton X-100. -
20x SSC: 3 M NaCl, 0.3 M Na3Citrate-2H20. Store at room temperature.-
Mounting media: 70% glycerol, 1mg/ml p-phenylenediamine (pH 9).

C. Working solutions
These solutions are prepared on the day of use.-
Formaldehyde fixative solution: 1X PBS, 0.08 M Hepes (pH 6.9), 1.6 mM MgSO4, 0.8 mM EGTA, 3.7% formaldehyde.-
Hybridization buffer for cDNA-derived probes: 100 µg/ml autoclaved salmon sperm DNA, 50 µg/ml heparin, 0.1% Tween 20, 50% formamide, 5 X SSC.
[ If you are using Wheaton jars that hold up to 20 slides in 150 ml, you will need a total of 1 liter of HYB buffer for the pre-HYB and post HYB washes:
Prepare 1 liter of HYB buffer omitting the DNA.
Aliquot HYB into 4 bottles as follows:
- 400ml in one bottle labeled HYB w/DNA. Add 4ml of 10mg/ml ssDNA to that aliquot.
- 360ml in one bottle labeled HYB wash
- 180ml in one bottle labeled 3:2. Add 120ml of PTw to that aliquot.
- 60ml in one bottle labeled 1:4. Add 240ml of Ptw to that aliquot.]-
Hybridization buffer for oligonucleotide probes: 100 µg/ml autoclaved salmon sperm DNA, 50 µg/ml heparin, 0.1% Tween 20, and appropriate concentrations of formamide and SSC as described in section III D.-
Hypochlorite solution: 1N NaOH, 1:10 dilution of commercial bleach. -
PBT: 1 x PBS, 0.1% BSA, 0.1% Triton X-100-
PTw: 1 x PBS, 0.1% Tween 20-
Staining solution: 100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH 9.5; 0.1% Tween 20; 1mM Levamisole. Levamisole is a potential inhibitor of endogenous phosphatases.-
TTBS: 150mM NaCl, 50mM Tris-HCl pH 7.8, 0.1% BSA, 0.1% Tween-20

E. Slides and other materials
- Carter's rubber cement (Dennison Stationary Products).-
Coverslips for freeze-cracking (No. 11/2; 24 x 50mm; Thomas Scientific Cat# 6663K94)-
Incubation dishes. Unless otherwise noted, all washes and incubations are done in Wheaton staining dishes (Thomas Cat# 8541-H15), which can hold 20 slides in 150 ml.-
Parafilm squares cut to 20 x 20 mm. -
Slides (75 x25mm) [Cel-Line Associates Inc. (tel: 1-800-662-0973), Cat# 10-2066, brown autoclavable coating]. These slides have 3 square wells (14x14mm) surrounded by a thin hydrophobic coating similar in thickness to a C. elegans embryo. This coating supports the coverslip during freeze-cracking and facilitates incubation with small volumes of staining solutions. Only the two outside wells are used. Wells are subbed with poly-lysine on the day of use: 50 µl of poly-lysine solution is allowed to settle on slides for 10-20 min; excess solution is wiped off and slides are baked at 60oC for 10 min.

II. PROCEDURE

A. Overview.
A mixed population of C. elegans embryos is attached to microscope slides, permeabilized by freezing and fixed with methanol and formaldehyde. Embryos are then incubated overnight with a digoxigenin-labelled single-stranded DNA probe, followed by extensive washes to remove excess probe. Fluorescent or enzyme-linked anti-digoxigenin antibodies are used to visualize the hybridized probe. The entire procedure requires approximately 1.5 days from harvest of embryos to probe visualization.
This protocol is designed for embryos, but can also be used for larvae/adults. Best results have been obtained when looking at RNAs expressed in the adult germline, but some somatic RNAs have also given nice patterns. Modifications necessary to work with larvae/adults are described in section D.

B. Probe synthesis

We have used two types of single-stranded DNA probes: probes derived from cloned cDNAs, and synthetic oligonucleotides. The cDNA-derived probes are used to detect mRNAs derived from specific genes. These probes are synthesised by multiple cycles of primer extension in the presence of digoxigenin dUTP, using a cloned cDNA as a template (eg. "asymmetric PCR", Patel and Goodman, 1992). The oligonucleotide probes are suitable for detecting abundant RNAs containing a defined sequence such as poly-A+ RNAs (using an oligo-dT probe) and SL1-bearing RNAs (using an anti-SL1 probe). These probes are end-labeled with terminal transferase and digoxigenin-ddUTP.

1. Protocol for preparation of single-stranded probes from cloned cDNA (from Patel and Goodman, 1992):
a) 2-5 µg of plasmid DNA containing the cDNA insert is linearized using an appropriate restriction enzyme. For antisense probes, a unique restriction site 5' to the insert is used. This digested DNA will be amplified using an antisense primer at the 3' end of the insert. For sense (control) probes, a unique restriction site 3' to the insert is used. This digested DNA will be amplified using a sense primer at the 5' end of the insert. (Inserts of up to 2kb are labeled efficiently).
b) Digested DNA is extracted once with phenol/chloroform, once with chloroform, precipitated with 3 volumes of 100% EtOH, and resuspended in TE at a final concentration of 100-200 µg/ml.
c) The following reagents are mixed in 0.5 ml eppendorf tube.
water 7.0 µl
10x Taq Buffer 2.5 µl
25 mM MgCl2 1.5 µl
10 x dNTP mix 5.0 µl
Primer * (30 ng/µl) 5.0 µl
Digested DNA (100-200 µg/ml) 2.0 µl
mineral oil 40 µl

~undefined For cDNAs cloned in Bluscript, we use the following primers (21mers):
"T3" = 5'- ACT AAA GGG AAC AAA AGC TGG -3'
"T7" = 5'- ACT CAC TAT AGG GCG AAT TGG -3'

d. Mixed reagents are boiled for 5 min, before adding 2.0 µl of a 1:8 dilution in water of 5 units/µl Taq polymerase stock (1.25 units total).
e. The labelling reaction is incubated for 35 thermal cycles as follows:
95°C for 45 seconds
55°C for 30 seconds (lower temperature for primers less than 20nt)
72°C for 1 minute
f. 75 µl of H20 is added to the reaction below the oil and 90 - 95 µl of the diluted reaction is transferred to a new tube.
g. 10 µl of 1M NaCl, 10 µg of glycogen, and 3 vols of 100% EtOH are added to the diluted reaction. After 30 min at -70°C, the reaction is centrifuged at 15,000 rpm for 10 min. The pellet is washed in 70% ethanol, dried, and resuspended in 300 µl of hybridization buffer.
h. The probe is boiled for 1-2 hours. This step reduces the length of the probe for efficient penetration of embryos.
e. Probe production is assayed using the following protocol. 1 µl of probe in hybridization buffer is mixed with 5 µl of 5 x SSC, boiled for 5 min, and cooled on ice. 1 µl of this mixture is spotted on a nitrocellulose strip. Several dilutions of a pre-labelled control DNA (1ng to 1pg / µl; Boeringher Mannheim) are also spotted for comparison. The strip is baked for 30 min in a vacuum oven at 80oC, washed once in 2 x SSC, twice in PBT, and blocked for 30 minutes in PBT. The strip is then incubated for 30-60 min with AP-anti DIG antibody diluted 1:2000 in PBT. After three 10 min washes in PBT, and two 5 min washes in staining solution, the strip is developed in staining solution containing 4.5 µl NBT/ml, 3.5 µl X-phosphate/ml. Spots should be visible within minutes. Spot intensities of the probe and control dilutions are compared to determine the concentration of the probe.
f. Probes can be stored at -20oC in hybridization buffer for several weeks.

2. Synthesis of oligonuceotide probes:
Synthetic oligonucleotides are end-labelled using terminal transferase and digoxigenin-ddUTP (Boeringher Mannheim sells an 3' end-labeling kit [Cat # 1362372]; we have used these reagents on gel-purified oligonucleotides). Labeled oligonucleotide probes are resuspended to 0.5 µg/ml in hybridization buffer. The percentage of formamide and concentration of SSC in the hybridization buffer are adjusted for each oligonucleotide to give a melting temperature (Tm) of 52oC, using the following formula (Davis et al., 1986):
Tm = 16.6 log[M] + 0.41[Pgc] + 81.5 - B/L - 0.65[Pf]
where M is the molar concentration of Na (maximum of 0.5), Pgc is the percent of G and C bases in the oligonucleotide, B is 675 for synthetic oligos up to 100 bases in length, L is the length of the probe in bases, and Pf is the percent concentration of formamide. A Tm of 52oC allows for efficient hybridization at 37oC.

C. Collection of Embryos
1. Worms are grown on a lawn of E. coli strain OP50 on NGM agar plates (100 x 15 mm). For the wild type strain N2, each plate is started with 20 adult hermaphrodites which are allowed to grow until all their progeny have started to lay eggs. Ten such plates yield enough embryos for approximately thirty individual wells. Chunks of agar on plates should be avoided, since these can interfere with the freeze-cracking step.
2. Gravid hermaphrodites and their laid eggs are washed off the plates in water, and collected into a 15 ml conical tube. After centrifugation (1700 rpm for a sufficient time to pellet embryos and adults, approximately 1min), most of the water above the pellet is removed.
3. The worm pellet is resuspended in 10 ml of hypochlorite solution, and incubated at room temperature for 3 minutes with some agitation. Worms are recovered by centrifugation and incubated in fresh hypochlorite solution for another 3 minutes (Incubation times may vary depending on the type of bleach used). Embryos are protected by their egg shell from hypochlorite digestion, while larvae and adults are dissolved by this treatment.
4. When carcasses of larvae and adults are no longer visible in the dissecting microscope, embryos are washed twice in 15 ml of PBS, before resuspending in a small volume of PBS (0.5 ml or less).

D. Permeabilization and fixation of embryos
1. Embryos are transferred to the polylysine-coated slides using a micro-pipet. (15 µl of embryos in PBS is sufficient to cover a 14 x 14 mm square well). Make sure the embryos are not forming clumps. Clumps can be separated by blowing air onto the embryos through a micropipet.
2. Embryos are overlayed with a glass coverslip and the slide is placed in a humidity chamber. Repeat for all slides

Modification of protocol for larvae and adults:
Wash worms in PBS, spin them down, resuspend in small volume of PBS, and spread 5ul containing 10 to 50 worms onto the polylysined 14x14mm well. Cover the worms with a coverslip making sure that the PBS spreads onto the painted part of the slide thus pulling the coverslip down. You can then press on the coverslip ever so lightly to burst open the hermaphrodites, so as to obtain best permeabilization of the germline and embryos. Proceed with freeze-cracking (step 3).

3. Slides are frozen by placing on an aluminum block that has been pre-cooled on dry ice. The coverslips are then quickly snapped off, and the slides are immediately immersed in 100% methanol at -20oC for 5 min.
3. Slides are transferred to 100% methanol at room temperature for 5 min and then rehydrated at room temperature as follows :
- One 1 min wash in 90% MeOH in H2O.
- One 1 min wash in 70% MeOH in PBS.
- One 1 min wash in 50% MeOH in PBS.
- Two 5 min washes in PTw
IF YOU DO NOT PLAN TO DO THE PROTEINASE K DIGESTION STEP, YOU CAN SKIP THESE LAST TWO PTw WASHES AND GO DIRECTLY FROM 50% MeOH TO THE FIXATIVE SOLUTION (step5).
4. At this point, a Proteinase K digestion step can be incorporated. Such a step may be necessary to increase the accessibility of low abundance messages expressed after the lima-bean stage (Pete Okkema, pers. communication). However, for earlier stages, proteinase K treatment is not recommended. Each batch of proteinase K needs to be titrated to determine proper incubation conditions. (Typically, a 15 min incubation in a 1 µg/ml solution in PTw at room temperature is sufficient if needed). Proteinase K digestion is stopped by incubating for 2 min in 2mg/ml glycine in PTw, followed by two 5 min washes in PTw. (If protease digestion is not necessary, proceed directly from step 3 to step 5).
5. Embryos are fixed by incubation at room temperature for 20 min in formaldehyde fixative solution.
6. To remove formaldehyde, embryos are washed extensively at room temperature as follows:
- Two 5 min washes in PTw.
- One 5 min wash in 2 mg/ml glycine in PTw.
- Three 5 min washes in PTw.

E. Prehybridization
1. Fixed embryos are incubated for 10 min in a 1:1 mix of hybridization buffer and PTw, followed by a 10 min incubation in undiluted hybridization buffer. (These incubations are done at room temperature). During this time, a separate 150 ml aliquot of hybridization buffer is heated in a boiling water bath for 10 min, and cooled on ice.
2. Embryos are pre-treated in this freshly-heated hybridization buffer for 1-2 hours at the temperature to be used for probe hybridization (48oC for cDNA-derived probes and 37oC for oligonucleotide probes).

F. Hybridization
1. cDNA-derived probes can be used undiluted (original 300µl, appr. 5 µg/ml) or up to nine-fold diluted depending on the abundance of the transcript. In general, the probe is diluted 3-fold for moderately abundant messages (eg. skn-1 mRNA) and up to nine-fold for very abundant messages (eg. unc-54 mRNA; lacZ mRNA derived from a multiple-copy array). Oligonucleotide probes are used at a concentration of 0.5 µg/ml.
2. The probe is boiled for 10 min then cooled on ice.
3. Slides are removed from the pre-hybridization buffer and excess buffer is wiped off while keeping the embryos wet.
4. 20 µl of diluted probe is added to each well. Each well is then covered with a square parafilm coverslip (20 x 20 mm) which is sealed onto the slide with rubber cement.
5. Slides are incubated in a sealed humidity chamber overnight at 48oC (for cDNA-derived probes) or 37oC (for oligonucleotide probes).
6. You can also incubate the post-HYB wash solutions (HYB wash, 3:2, 1:4 and PTw) in the same incubator overnight.

E. Post-hybridization washes
After hybridization, parafilm coverslips are removed with forceps while keeping the slides submerged in hybridization solution. Embryos are then washed with gentle agitation (eg. 40-60 rpm in shaker with 17 cm radius) as follows:
1. Washes for cDNA-derived probes: (HYB solution used in washes does not require ssDNA)
- Two 15 min washes in hybridization solution at 48oC.
- Two 15 min washes in 3 parts hybridization solution / 2 parts PTw at 48oC.
- Two 15 min washes in 1 part hybridization solution / 4 parts PTw at 48oC.
- Two 15 min washes in PTw at 48oC.
- Two 20 min washes in PBT at room temperature.
2. Washes for oligonucleotide probes:
- One 10 min wash in hybridization solution at 37oC.
- Two 10 min washes in TTBS at room temperature.

F. Probe Detection
Two types of labels are available for detection: marker enzymes such as alkaline-phosphatase (AP) and fluorescent tags such as rhodamine or fluorescein. AP-mediated detection is highly sensitive but results in a signal with limited sub-cellular resolution. In contrast, fluorescent detection is generally less sensitive but allows more defined sub-cellular resolution. For these reasons, we prefer to use AP-mediated detection to identify cells expressing specific mRNAs, and fluorescent-detection to determine the sub-cellular localization of relatively abundant RNAs.

1. AP-mediated detection:
a. 30 µl of the diluted AP-anti-DIG antibody conjugate (1: 2500 in PBT, 0.3 units/ml) is applied to each well. Each well is then covered with a square parafilm coverslip and incubated in a humidity chamber for 2 hours at room temperature.
b. Embryos are washed 4 times for 10 min in PBT. (If an oligonucleotide probe was used, embryos are washed twice for 10 min in TTBS instead.)
c. Embryos are incubated twice for 5 min in freshly-made staining solution.
d. 30 µl of staining solution with 4.5 µl NBT/ml, 3.5 µl X-phosphate/ml and 1 µg DAPI/ml is applied to each well. Slides are kept in the dark during the color reaction. The signal should appear after 20 min to one hour depending on the probe. The color reaction is monitored under the microscope to avoid background, and is stopped by washing embryos twice in PBS.
e. Embryos are mounted in 5-10 µl of a 70% glycerol solution and covered with a glass coverslip.

2. Fluorescence detection.
a. 30 µl of Rhodamine-anti-DIG antibody conjugate (0.2 mg/ml with 1µg DAPI/ml) is applied to each well. Each well is then covered with a square parafilm coverslip and incubated in a humidity chamber for 2 hours at room temperature.
b. Embryos are then washed 4 times for 10 min in PBT. (If an oligonucleotide probe was used, embryos are washed twice for 10 min in TTBS instead.) For increased sensitivity, a secondary antibody can also be used.
e. Embryos are mounted in 5-10 µl of mounting media and covered with a glass coverslip.

III. DOUBLE-LABELLING

To facilitate the identification of cells in whole mount embryos, it is often useful to label specific cells using antibodies or other markers. Here, we describe the use of an anti-P granule antibody to label the P cells (P1-P4) and their germ cell descendants (Z2 and Z3) (Strome and Wood, 1982) in embryos that have been subjected to the in situ hybridization protocol described above. In theory, any antibody could be used in a similar manner, provided that its epitope is not destroyed by the hybridization procedure.

1. After completion of the detection step but before mounting the embryos in glycerol, the embryos are subjected to three 10 min washes in TTBS.
2. Embryos are incubated for 2 hours at room temperature with an FSE-labeled anti-P granule antibody (diluted 1:400 in TTBS; we use FSE-conjugated OIC1D4 from Janet Paulsen and Susan Strome).
3. Embryos are washed twice for five minutes in TTBS, before mounting in 5 µl of mounting media. P granule staining should be seen in most embryos. Strong alkaline phosphatase staining can quench the fluorescence of the anti-P granule antibody conjugate.

IV. INTERPRETATION

A. Generality of the technique
We have used this protocol to analyse the expression pattern of 21 genes expressed during embryogenesis (Seydoux and Fire, 1994). We find that the protocol allows the visualization of RNA in well-preserved embryos from the one-cell stage to the pretzel stage (see Fig. 1). In general, RNAs are detected in the cytoplasm of cells, with the exception of embryonically transcribed RNAs which can sometimes be detected in nuclei when they are first transcribed in early blastomeres. Low abundance RNAs expressed after the lima bean-stage can be difficult to detect; proteinase K digestion of embryos prior to hybridization can be helpful in such cases (Pete Okkema, pers. comm.). Although the protocol was developed to detect RNAs in embryos, preliminary results suggest that it is also be applicable for detecting RNAs in larvae and adults (G. S. and A. F., unpublished data).

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.

Most of this protocol is derived from methods described in the following references:

Patel, N. H. and Goodman, C. S. (1992). Preparation of digoxigenin-labeled single-stranded DNA probes. In Non-radioactive Labeling and Detection of Biomolecules. (ed. C. Kessler). Springer-Verlag, Berlin.

Tautz, D. and Pfeifle, C. (1989). A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback . Chromosoma 98, 81-85.