Protocols for large scale in situ hybridization on C . elegans larvae *

Copyright: © 2006 Tomoko Motohashi et al. 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: ykohara@lab.nig.ac or isugiura@lab.nig.ac.jp. Protocols for large scale in situ hybridization on C. elegans larvae* Tomoko Motohashi, Hiroaki Tabara, Yuji Kohara, Genome Biology Lab Center for Genetic Resource Information, National Institute of Genetics, Mishima 411–8540, Japan

6. Aspirate the sup.7. Measure the packed volume of the worms.
• If it is 2-3 ml, add DW to 10 ml.
• If it is 3-5 ml, add DW to 12.5 ml.
• If it is larger than 5 ml, divide the worms into multiple tubes.
8. Add equal volume of 2X alkaline-bleach solution and mix gently.NaClO 3 ml 5M KOH 2.5 ml DW 19.5 ml 9. Lay the tube down, monitoring the breakage of the worms under a dissecting microscope.

2X alkaline-bleach solution
10.When about 30% of the worms begin to break apart (usually 5-10 min later), load the suspension into a 50 ml disposable syringe.
11. Force it out through a needle (23G6) into a 50 ml Falcon tube.
12. Filtrate the suspension through a 50 mm nylon mesh, and wash the debris with M9 on the mesh to recover the trapped eggs.
13. Transfer the filtrate into 50 ml Falcon tubes.
14. Collect and wash eggs by centrifugation at 3000rpm for 1 min once and at 2000rpm for 1 min twice at 4°C.

15.
Transfer the eggs into 15 ml Falcon tube and centrifuge at 2000rpm for 1min at 4°C.
16. Measure the packed volume of the eggs.

Cultivation for preparation of staged worms
To cover all larval stages, synchronization at L1 is not performed.We usually cultivate worms at 20°C.After appropriate time, collect worms by: for L3 adults, sieving through 50µl nylon mesh and washing off with M9 into a 50ml Falcon tube.For L1-L2, centrifugation at 2000rpm and 4°C for 1 min.
4. Transfer the worms into 2 ml eppendorf tubes at 200µl (packed volume) worms per tube.
Protocols for large scale in situ hybridization on C. elegans larvae 6.Let the tubes stand for 30 sec to settle the worms down to the bottom.

Fixation
4. Check the density of the worms by counting worms in an aliquot of the suspension under a dissecting microscope.
5. Allow the worms to stick to slides as follows: 1. Place poly-L-lysine coated 8 well test slides on the top of an aluminum block pre-cooled on ice.
2. Dispense ice-cold PBS to individual wells at 30µl/well.
3. Dispense the rehydrated worms to individual wells at 5µl/well as follows: • L1-L2 is in the wells #1 and 5 • L2-L3 is in the wells #2 and 6 • L3-L4 is in the wells #3 and 7 • L4-adult is in the wells #4 and 8 4. Let stand for 5 min to settle the worms to the bottom.
6. Fix the worms as follows: 1. Soak the slides in MeOH pre-cooled at 4°C by arranging the slides in a stainless steel holder (15 slides/holder) that is placed in the MeOH.

Hybridization
1. Take the fixed slides, arrange in a stainless holder and immersed in EtOH.f.Cover the slides with the upper block and rock the complex.

Start of hybridization:
a. Apply all of the heat denatured probes using a 4-channel pipette.
b. Add 100µl of mineral oil per well.
c. Seal the holes of the apparatus with microtiter plate sealing tape.
d. Place the hybridization apparatus in a air-tight box.

3 .
Change the buffer to PBS (4°C), and rotate the tubes for 2 min at r.t. 4. Repeat step 3 once.5. ProteinaseK digestion: a. Add PBT (at 22°C) to total 1ml.b.Add 5µl of ProteinaseK (20mg/ml).c.Rotate the tubes for 12 min at 22°C. 6. Change the buffer to Glycine in PBT (at 4°C) and rotate the tubes for 2 min at r.t.

7 .
Change the buffer to PBS and rotate for 2 min at r.t. 8. Repeat step 7 twice.9. Fixation with Dent: Change the buffer to Dent (MeOH:DMSO = 8:2) pre-cooled at -20°C, and rotate for 5 min in cold room.

2 . 11 .
Let stand for 5 min.Protocols for large scale in situ hybridization on C. elegans larvae 3. Soak the holder with the slides in the following series of solution at 4°C in cold room: MeOH:formaldehyde in hepes-PBS = 760µl of 20mg/ml of ProteinaseK in 180ml of PBT pre-warmed at 37°C (final conc.µg/ml).b.Mix well by stirring.c.Transfer into a vat that fits the slide holder.d.Soak the holder containing the slides in the ProteinaseK solution.e. Incubate at 37°C for 30 min.8. Transfer the holder in glycine in PBT pre-cooled at 4°C and let stand for 2 min to stop the digestion.9. Acetylation a. Soak in 0.1% Triethanol amine for 2 min at r.t.b.Soak in 0.05% Acetic anhydride in Triethanol amine for 10 min.10.Dehydrate the specimen by soaking the holder in the following series of solution at r.t.: Store the slides in EtOH at −80°C.

2 . 4 .
Rehydrate the specimen by soaking the holder in the following series of solutions: formamide, 5XSSC, 100µ/ml heparin, 0.1% Tween:PBT = 1:1 10 min 50% formamide, 5XSSC, 100µ/ml heparin, 0.1% Tween 10 min Protocols for large scale in situ hybridization on C. elegans larvae 3. Prehybridization a.Take out the slides using forceps, wipe off the outside of the wells and draw a rectangle surrounding the 8 wells using a IMMUNO pen.b.Pour 250µl of hybridization solution (heat-denatured at 99°C for 10 min.and quickly chilled on ice-water for 5 min) inside the rectangle.c.Placed the slides in a moisture box.d.Place the moisture box in an oven at 48°C for 1hr.Heat denature probes as follows: a. Dispense 9µl probe solution/well into 4 contiguous wells (e.g., A1-A4), since one probe is applied to 4 wells (for 4 different larval stages).b.Dispense 41µl of hybridization solution/well and mix by pipetting.c.Seal the plate using GeNunc Tape and centrifuge.d.Place the plate on a heated block at 99°C for 10 min and quickly chill on ice for 5 min.5. Assembling of hybridization apparatus (S&S 96 well dot blotting apparatus): a. Place a silicon sheet (1 mm thick) on the top of the lower block.b.Clean up the surface of the silicon sheet with EtOH.c. Apply O-rings to the holes at the 4 corners and the holes used for hybridization of the upper 96-hole block.d.Take out the pre-hybridized slides, drain off the hybridization solution by tapping on the top of paper towel.e.Quickly arrange 4 slides at the fixed positions on the silicon sheet on the lower block.

2.1. Primary fixation of worms
Protocols for large scale in situ hybridization on C. elegans larvae 11.Dehydration: Change the buffer and rotate the tubes at r.t. as follows: 10. Rehydration: Change the buffer and rotate the tubes as follows:(The amounts of worms allows hybridization with 120 different probes.)2. Rehydration: Change the buffer and rotate the tubes at r.t. as follows: