Embryo series courtesy of Einhard SchierenbergTable of Contents
Make the following reaction mix (total 10 μl):
| distilled water | 4.4 μl |
| anchored oligo dT primers* | 3.0 μl |
| 10 × Taq buffer | 1 μl |
| Taq polymerase | 0.1 μl |
| 10 × DIG-dUTP/dNTP mix** | 1.5 μl |
|
cDNA insert (> 3.5 ng/ μl) amplified from yk clone using T7/T3 primers |
0.6 μl |
|
* anchored oligo dT primers: 5.3 μM (dT)17dG/5.3 μM (dT)17dC/21.3 μM (dT)17dA (This is to avoid the effect of poly-A stretch. Other vector primers may be used.) ** 10 × DIG-dUTP/dNTP mix: 0.35 mM DIG-dUTP/0.65 mM dTTP/1mM each d(A, G, C)TP |
Subject to thermal cycling:
hot start at 95 °C for 45 sec
95 °C × 15 sec
45 °C × 1 min
72 °C × 1 min
50 cycles
Add 10 μl of 10 mM EDTA
Pass through G-50 spin colum chromatography (ca. 250 μl bed volume)
Add 5 μl of TSE
Make the following reaction mix (total 25 μl) on ice:
| The G-50 elutate | 20 μl |
| 10 mg/ml Salmon sperm DNA | 1 μl |
| distilled water | 0.5 μl |
| 10 × DNase buffer* | 2.5 μl |
| DNaseI (16 μg/ml)** | 1 μl |
|
* 10 × DNase buffer: 0.5 M TrisHCl pH 7.5, 0.1 M MgCl2 ** Dilute stock solution (1 mg/ml) with 0.1 M NaCl. (Note : Best size of probes is about 100 bases. Longer probes may cause high background. The concentrations of the enzyme should be optimized by pilot experiments.) |
Incubate at 37 °C for 30 min
Transfer on ice
Add 5 μl of 0.1 M EDTA
Heat at 75 °C for 5 min
Check the size by alkaline agarose gel electrophoresis and DIG detection, if necessary
Store frozen
Take siliconized 1.5 ml eppendorf tubes
Place about 100 μl of distilled water on the (inside) top of the lids
Pick and transfer 40-50 gravid worms into the water. If you need very late stage embryos:
Add 50 μl of suspension of E. coli OP50 in S-basal
Cover the lid with the body of the tube
Let stand at 20 °C overnight
Spin down the worms.
Add equal volume of 2 × alkaline-bleach solution and mix well
Leave at r.t. for 10 min to dissolve the adult bodies
Add 1 ml of M9 buffer at 4 °C
Centrifuge at 2500 rpm for 30 sec at 4 °C in a swing rotor
Remove the sup carefully, leaving about 100 μl of the sup to avoid removing the embryos
Repeat steps 7-9 three more times
Add an equal volume of 3 mg/ml chitinase
Mix and incubate at r.t. for 3 min
Spin at 2500 rpm for 30 sec at 4 °C
Reduce the volume to about 50 μl
Transfer the embryos to a poly-L-lysine coated 3-well slide using a siliconized pipette tip
Add a half volume of 4% gelatin, 2% BSA, and mix gently by pipeting
Let stand for several minutes to allow the embryos settled down to the bottom
Cover with a cover slip (24 × 40 mm)
Place it on the top of a dry ice block
Freeze for 7 min at -70 °C
Peel off the cover slip quickly
Soak the slide in methanol cooled at -20 °C for 5 min
Rehydrate by soaking the slide in the series of the following solutions pre-cooled at 4 °C:
| methanol | for 5 min |
| methanol:formaldehyde-Hepes-PBS* = 35:15 | for 2 min |
| methanol:formaldehyde-Hepes-PBS* = 25:25 | for 2 min |
| methanol:formaldehyde-Hepes-PBS* = 15:35 | for 2 min |
| formaldehyde-Hepes-PBS* | for 20 min |
Dehydrate by soaking the slide in the series of the following solutions at r.t.:
Store in ethanol at −20 °C.
Get a plenty of worms from a mixed stage population
Wash the worms 2 times with M9 buffer
Collect L1-L3 by sieving through 50 μm Nylon mesh
Allow the collected worms to grow to young adults in liquid culture
Take 1 ml packed worms from the culture, which will give 8-15 slides for in situ
Resuspend the worms in 4 ml water in a 15 ml Falcon tube (clear type)
Add 5 ml of 2 × alkali-bleach solution, mix well and let stand for 10 min
Force the worms out through a 23-gauge needle onto nylon mesh
Collect embryos by spinning the filtrate at 800 × g using a swing rotor
Wash the embryos 4 times with M9 and transferred into a siliconized eppendorf tube
Take 100 μl (packed volume) of the embryos and adjust the volume to 200 μl with M9
Add 200 μl of yatalase (15 mg/ml in 0.3 M mannitol) and vigourously shake for 75 sec
Wash the embryos 3 times with EH buffer (Embryo Handling buffer)
Wash the embryos with Basal EH buffer
Resuspend the embryos in 1 ml of Basal EH buffer. (Note: For success, it is desired that 20-30% of embryos are devitellinized at this step)
Place 30 μl/well of Basal EH buffer onto each well of poly-L-lysine coated 8-well slides
Dispense 5 μl/well of the embryo suspension into the buffer at each well
Let stand for 10 min at 4 °C to settle the embryos to the bottom
Remove the buffer, and immediately immerse in methanol at -20 °C for 5 min
Rehydrate the embryos by immersing the slides in the follwoing series at 4 °C. The solutions must be pre-cooled at 4 °C.
Dehydrate the embryos by immersing the slides in the following series at r.t.
Store in ethanol at -20 °C.
Rehydrate the embryos by immersing the follwoing ethanol series:
Wash the slides once by immersing in PBT for 5 min. *For late stage embryos, additional HCl treatment is effective, which can cut glycosid bonds of the proteoglycan that appear on late stage embryos.
a) Immerse the slides in 0.2 N HCl for 20 min at r.t.
b) Wash the slides 2 times in PBT for 5 min
Immerse the slides in proteinase K (10 μg/ml in PBT) and incubate at r.t. for 11 min
Stop the digestion by immersing the slides in 2 mg/ml glycine in PBT for 2 min
Wash the slides twice by immersing them in PBT for 2 min each
Refix by immersing the slides in 3.7% formaldehyde in hepes-PBS at r.t. for 50 min
Wash the slides twice in PBT for 5 min each
Immerse the slides in 2 mg/ml glycine in PBT at r.t. for 5 min
Wash the slides in PBT for 5 min
Immerse the slides in the following series of mixtures;
Wipe off the slides
Draw a rectangle surrounding the sample wells using a IMMUNO pen to make a ridge
Add 250 μl of heat denatured (at 99 °C for 10 min and quickly chilled for 5 min) hybridization solution for each 8-well slide
Place the slide in a moist chamber containing a paper towel wetted with 50% formamide, 5 × SSC. (No need to use coverslips)
Incubate at 48 °C for 1 hr
Add 50 μl of heat-denatured DNA probes for each slide. (The final concentrations of probes is about 0.06 μg/ml)
Cover the slide with a parafilm coverslip to reduce evaporation
Incubate the slides at 48 °C overnight in the moist chamber
Wash the slides in the following series of washing solutins at 48 °C with slight agitation
Wash the slides twice in PBT for 5 min at r.t. to remove CHAPS.
Incubate the slides in PBtr (PBS, 0.1% Triton-X100, 0.1% BSA, 0.01% NaN3) for 1.5 hr at r.t.
Cover the embryos with 250 μl of anti-DIG conjugate (dilute 1:2500)/8-well slide
Incubate for 2 hrs at r.t. in a moist chamber. (no need to use coverslips)
Wash the slides with PBtr 4 times with slight agitation
Wash the slides with the staining buffer (see reagents) twice for 5 min each at r.t.
Color development
a) Mix 180 μl of NBT and 140 μl of BCIP in 40 ml of staining buffer.
b) Immerse the slides in the mixture for 1 hr at 22 °C in the dark, monitoring the extent of the staining.
Wash the slides three times with PBS, 20 mM EDTA to stop the reaction.
If necessary, incubate the slides in 1 μg/ml DAPI in Tris buffer at 4 °C for 30 min.
Add about 90 μl of “MOUNT-QUICK AQUEOUS” onto the embryos on the slide
Cover with a coverslip
Let stand one day to dry up
Seal up the edge of the coverslip using nail varnish
Dehydrate with the following ethanol series:
Wash once with ethanol:Histo-Clear (National Diagnostics) = 1:1
Wash once with Histo-Clear
Add drops of Mount-Quick onto the embryos and cover with a coverslip
Leave the slide at 40 °C for several hours
(Note : Hybridization signals by this method tend to be weaker than those by other methods and to diffuse, but preservation of morphology is better than other methods)
S-basal
| NaCl | 69 g |
| 1M K-PO4 (pH6) | 100 ml |
| cholesterol (5 mg/ml in EtOH) | 2 ml |
| Add DW to total 2 liter and autoclave | |
2 × alkali-bleach solution
PBS
PBT
PBS + 0.1% Tween 20
EH buffer (Embryo Handling buffer)
| mannitol | 0.3 M |
| Hepes pH 7.2 | 50 mM |
| NaCl | 10 mM |
| MgCl2 | 10 mM |
| EGTA | 0.04% |
| NH4NO3 | 2 mM |
| gelatin | 0.1% |
| DTT | 2 mM |
Basal EH buffer
(= EH buffer without EGTA, NH4NO3, gelatin and DTT)
Glycine in PBT
3.7% Formaldehyde in hepes-PBS
hepes buffer*:formalin:10 × PBS = 8:1:1
*hepes buffer
Hybridization solution
| deionized formamide | 50% |
| SSC (pH7, autoclaved) | 5× |
| sonicated salmon testis DNA | 100 μg/ml |
| yeast tRNA | 100 μg/ml |
| heparin | 100 μg/ml |
| Tween 20 | 0.1% |
yatalase (15 mg/ml) and chitinase (1 mg protein/ml = 5 mg crude/ml)
Dissolve powder of yatalase (TAKARA No.T017) or chitinase (SIGMA No. C-6137) in 0.3 M mannitol, 50 mM Hepes pH 7.2, 10 mM NaCl, 10 mM MgCl2, 2 mM DTT
Filtrate through a 0.45 μm syringe filter.
store at -20 °C.
Digoxigenin-11-dUTP (Roche 1570013)
PBT
0.1% Tween-20 in PBS (0.01% DEPC treated)
PBtr
0.1% BSA (Fraction V), 0.1% Triton X-100 in PBS proteinase K stock solution
20 mg Proteinase K (Roche 30U/mg)/ml water
Staining buffer (Alkaline phosphatase reaction buffer)
poly-L-lysine coated slides
Immerse glass slides in solution of kitchen detergent for 20 min
Wash in tap water for 1 hr
Wash in ion-exchanged water
Autoclave and dry at 80 °C
Drop poly-L-lysine solution (SIGMA P-8920) onto individual wells of the slides
Leave for 25 min
Aspirate off the excess solution (only when 3-well slides are used)
Dry at 65 °C for 1 hr
Parafilm coverslips
Dribble beads of rubber cement along the edge of square pieces of parafilm
Dry briefly at 35-40 °C
*Edited by Ikuko Muramatsu, Masumi Obara, Wakako Shimizu, Aya Hamakawa, and Yuji Kohara. Last revised April 27, 2005. Published May 19, 2015. This chapter should be cited as: Motohashi T., Hirono K., and Kohara Y. In situ hybridization on whole mount embryos of C. elegans (May 19, 2015), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.176.1, http://www.wormbook.org.
Copyright: © 2015 Tomoko Motohashi, Keiko Hirono and Yuji Kohara. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
§To whom correspondence should be addressed: Email: Ikuko Muramatsu, [email protected], Yuji Kohara, [email protected]
All WormBook content, except where otherwise noted, is licensed under a Creative Commons Attribution License.
