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

Tomoko Motohashi, Hiroaki Tabara and Yuji Kohara
Genome Biology Lab
Center for Genetic Resource Information
National Institute of Genetics
Mishima 411-8540, Japan

Corrected by Ikuko Muramatsu, Masumi Obara, Wakako Shimizu, Aya Hamakawa and Yuji Kohara (April 27, 2005)
Correspondence: Ikuko Muramatsu <>, Yuji Kohara <>


  1. Preparation of staged worms
    1. Preparation of clean eggs
    2. Cultivation for preparation of staged worms
  2. Fixation
    1. Primary fixation of worms
    2. Fixation of worms onto slides
  3. Hybridization and detection
    1. Hybridization
    2. Washing
    3. Staining by enzyme reaction
  4. Reagents

A. Preparation of staged worms

I. Preparation of clean eggs

  1. Sieve a liquid culture containing a lot of gravid worms through nylon mesh (50 μm).
  2. Clean up the collected worms thoroughly with DW on the nylon mesh.
  3. Wash off the worms on the mesh with DW into a beaker.
  4. Transfer the worms into a 50ml centrifuge tube.
  5. Wash the worms by centrifugation (2000rpm for 1min at 4oC).
  6. Aspirate the sup.
  7. Measure the packed volume of the worms.
  8. Add equal volume of 2 x alkaline-bleach solution (*) and mix gently.
    (*) 2 x alkaline-bleach solution:
    NaClO3 ml
    5M KOH2.5 ml
    DW19.5 ml
  9. Lay the tube down, monitoring the breakage of the worms under a dissecting microscope.
  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 μm 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 4oC.
  15. Transfer the eggs into 15 ml Falcon tube and centrifuge at 2000rpm for 1min at 4oC.
  16. Measure the packed volume of the eggs.

II. Cultivation for preparation of staged worms

To cover all larval stages, synchronization at L1 is not performed. We usually cultivate worms at 20oC.
  1. Mix the eggs, S-basal and E.coli OP-50 suspension in a new 1l flask as follows;
    eggsS-basalOP-50 Collect afterExpected vol of worms
    For L1-L2 100 μl200 ml30 ml20-24 hrs 150-200 μl
    For L2-L3 100 μl200 ml80 ml48 hrs 500 μl
    For L3-L4 50 μl200 ml50 ml60 hrs 500 μl
    For L4-adult 50 μl200 ml90 ml70-72 hrs 2.5 ml
  2. After appropriate time, collect worms by:
  3. Wash the worms with M9 by centrifugation (2000rpm, 4oC for 1 min).
  4. Transfer the worms into 2 ml eppendorf tubes at 200 μl (packed volume) worms per tube.
  5. Centrifuge the tubes at 3500rpm for 10sec at 4oC.
  6. Let the tubes stand for 30 sec to settle the worms down to the bottom.
  7. Remove the sup using aspirator.
    (This procedure will be used for changing buffer in the subsequent steps.)

B. Fixation

I. Primary fixation of worms

  1. Add 10mM DTT, 0.1% Tween-20 in 1 x BO3(pH9) equilibrated at 22oC.
  2. Rotate the tubes for 20 min at 22oC.
  3. Change the buffer to PBS (4oC), and rotate the tubes for 2 min at r.t.
  4. Repeat 3. once.
  5. ProteinaseK digestion:
    1. Add PBT (at 22oC) to total 1ml.
    2. Add 5 μl of ProteinaseK (20mg/ml).
    3. Rotate the tubes for 12 min at 22oC.
  6. Change the buffer to Glycine in PBT (at 4oC) and rotate the tubes for 2 min at r.t.
  7. Change the buffer to PBS and rotate for 2min at r.t.
  8. Repeat 7. twice.
  9. Fixation with Dent:
    Change the buffer to Dent (MeOH:DMSO = 8:2) pre-cooled at -20oC, and rotate for 5 min in cold room.
  10. Rehydration:
    Change the buffer and rotate the tubes as follows;
    MeOHat 4oCfor 5 min
    MeOH:0.2N HCl = 1:1at 4oCfor 10 min
    PBSat 4oCfor 2 min
    PBSat 22oCfor 5 min
    10mM DTT in 1 x BO3(pH9)at 22oC for 10 min
    1 x BO3(pH9)at 22oC for 3 min, 2 min, 2 min
    (3 times)
    0.6% H2O2 in 1 x BO3 at 22oCfor 10 min
    (Add 1 x BO3 to total 1ml and then add 20 μl of 30% H2O2)
    PBSat 22oCfor 2 min 3 times
    3.7% formaldehyde in hepes-PBSat 22oC for 2hrs
    (Freshly prepared and stored in a refrigerator until use.)
  11. Dehydration:
    Change the buffer and rotate the tubes at r.t. as follows;
    EtOH:PBS = 3:75 min
    EtOH:PBS = 1:15 min
    EtOH:PBS = 7:35 min
    EtOH5 min twice
  12. Store the fixed worms at -20oC in EtOH

II. Fixation of worms onto slides

  1. Resuspend the fixed worms (stored in EtOH at -20oC) and quickly transfer the following volume (variable depending on the sample worms) of the suspension into siliconized 2 ml eppendorf tubes;
    L1-L2ca. 200 μl/tube
    L2-L3ca. 300 μl/tube
    L3-L4ca. 900 μl/tube
    L4-adultca. 1100 μl/tube
    (The amounts of worms allows hybridization with 120 different probes.)
  2. Rehydration:
    Change the buffer and rotate the tubes at r.t. as follows;
    EtOH:PBS = 7:3for 5min
    EtOH:PBS = 1:1for 5min
    EtOH:PBS = 3:7for 5min
  3. Wash with PBT for 5 min x 3 times and resuspend in about 700 μl of PBT.
  4. Check the density of the worms by counting worms in an aliquot of the suspension under a dissecting microscope.
  5. Allow the worms 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 30 μl/well.
    3. Dispense the rehydrated worms to individual wells at 5 μl/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 and8
    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 4oC by arranging the slides in a stainless steel holder (15 slides/holder) that is placed in the MeOH.
    2. Let stand for 5 min.
    3. Soak the holder with the slides in the following series of solution at 4oC in cold room;
      MeOH:formaldehyde in hepes-PBS = 7:32min
      MeOH:formaldehyde in hepes-PBS = 1:12min
      MeOH:formaldehyde in hepes-PBS = 3:72min
      3.7% formaldehyde in hepes-PBS60min
      PBT5min x 5 times at r.t.
  7. ProteinaseK digestion:
    1. Add 60 μl of 20mg/ml of ProteinaseK in 180ml of PBT pre-warmed at 37oC .
      (final conc. 6.7 μg/ml).
    2. Mix well by stirring.
    3. Transfer into a vat that fits the slide holder.
    4. Soak the holder containing the slides in the ProteinaseK solution.
    5. Incubate at 37oC for 30 min.
  8. Transfer the holder in glycine in PBT pre-cooled at 4oC and let stand for 2 min to stop the digestion.
  9. Acetylation
    1. Soak in 0.1% Triethanol amine for 2 min at r.t.
    2. 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.;
    formaldehyde in hepes-PBS20min
    EtOH:PBS = 3:75min
    EtOH:PBS = 1:15min
    EtOH:PBS = 7:35min
    EtOH5min twice
  11. Store the slides in EtOH at -80oC.

C. Hybridization and detection

I. Hybridization

  1. Take the fixed slides, arrange in a stainless holder and immersed in EtOH.
  2. Rehydrate the specimen by soaking the holder into the following series of solution;
    EtOH:PBS = 7:3for 5 min
    EtOH:PBS = 1:1for 5 min
    EtOH:PBS = 3:7for 5 min
    PBTfor 5 min
    50% formamide, 5XSSC, 100 μg/ml heparin, 0.1% Tween:PBT = 1:1for 10 min
    50% formamide, 5XSSC, 100 μg/ml heparin, 0.1% Tween for 10 min
  3. Prehybridization:
    1. 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.
    2. Pour 250 μl of hybridization solution (heat-denatured at 99oC for 10 min and quickly chilled on ice-water for 5 min) inside the rectangle.
    3. Place the slides in a moisture box.
    4. Place the moisture box in an oven at 48oC for 1hr.
  4. Heat denature probes as follows;
    1. Dispense 9 μl probe solution/well into 4 contiguous wells (e.g. A1-A4), since one probe is applied to 4 wells (for 4 different larval stages).
    2. Dispense 41 μl of hybridization solution/well and mix by pipetting.
    3. Seal the plate using GeNunc Tape and centrifuge.
    4. Place the plate on a heated block at 99oC for 10 min and quickly chill on ice for 5 min.
  5. Assembling of hybridization apparatus (S&S 96 well dot blotting apparatus):
    1. Place a silicon sheet (1 mm thick) on the top of the lower block.
    2. Clean up the surface of the silicon sheet with EtOH.
    3. Apply O-rings to the holes at the 4 corners and the holes used for hybridization of the upper 96-hole block.
    4. Take out the pre-hybridized slides, drain off the hybridization solution by tapping on the top of paper towel.
    5. Quickly arrange 4 slides at the fixed positions on the silicon sheet on the lower block.
    6. Cover the slides with the upper block and rock the complex.
  6. Start of hybridization:
    1. Apply all of the heat denatured probes using a 4-channel pipette.
    2. Add 100 μl of mineral oil per well.
    3. Seal the holes of the apparatus with microtiter plate sealing tape.
    4. Place the hybridization apparatus in a air-tight box.
    5. Incubate at 48oC overnight.

II. Washing

  1. Pre-warm the following solution
  2. Dispense solution-1 into the hybridization holes (to dilute the probes).
  3. Discard the solution in the holes by decantation.
  4. Disassemble the apparatus, take the slides and arrange them in a holder soaked in solution-1.
  5. Shake for 2 min in a 48oC incubator.
  6. Transfer the holder containing the slides into a new vat containing solution-1 and shake for 10 min in the 48oC incubator.
    Repeat once.
  7. Transfer the holder into a new vat containing solution-2 and shake for 20 min in the 48oC incubator.
    Repeat 3 times.
  8. Transfer the holder into a new vat containing 1xPBT and shake for 5 min at r.t..
    Repeat once.
You may store the slides in 1xPBT at 4oC overnight.

III. Staining by enzyme reaction

  1. Transfer the holder containing the slides into a vat containing PBtr (PBS, 0.1% Triton-X100, 0.1% BSA, 0.01% NaN3) and shake for 1.5 hr at r.t..
  2. Take individual slides, remove the solution outside the wells and overwrite the rectangle using a IMMUNO pen.
  3. Apply 250 μl of diluted anti-DIG antibody solution (diluted 1:2500 with PBtr) per slide.
  4. Place the slides in a moist box.
  5. Incubate for 2 hrs at r.t., or overnight at 4oC in the dark.
  6. Transfer the slides into a vat containing PBtr (PBS, 0.1% Triton-X100, 0.1% BSA, 0.01% NaN3) and shake for 10 min at r.t..
    Repeat 3 times.
  7. Soak the slides in stain buffer (100mM NaCl, 5mM MgCl2, 100mM TrisHCl pH9.5, 0.1% Tween, 1mM levamisol) and shake for 5 min at r.t.
    Repeat once.
  8. Arrange the slides in glass vats (max. 8 slides per vat) containing the stain buffer.
  9. Prepare the coloring solution by mixing 40ml of the stain buffer (at 22oC), 180 μl of NBT and 140 μl of BCIP.
  10. Decant the stain buffer from the glass vat preventing the coming out of the slides, and add the coloring solution into the vat.
  11. Incubate for 1hr 15 min in a 22oC incubator.
  12. Wash the slides 3 times with PBS, 20mM EDTA to terminate the coloring reaction.
  13. The slides can be stored in PBS, 20mM EDTA overnight at 4oC.
  14. Mount the slides using glycerol solution.
  15. Observe on a Microscope equipped with Nomarski optics.


KH2PO43 g
Na2HPO46 g
1M MgSO41 ml
Add DW to total 1 liter and autoclave

NaCl11.69 g
1M K-PO4 (pH6)100 ml
cholesterol (5 mg/ml in EtOH)2 ml
Add DW to total 2 liter and autoclave

40 x BO3 (pH9)
H3BO3 1M
Adjust pH to 9.0 using NaOH and autoclave

NaCl137 mM
KCl2.7 mM
Na2HPO44.3 mM
KH2PO41.5 mM
Adjust pH to 7.2 and autoclave

PBS + 0.1%Tween 20

Glycine in PBT
Glycine 2 mg/ml in PBS
Autoclave, then add 0.1% Tween 20

3.7% formaldehyde in hepes-PBS
hepes buffer(*) : formalin : 10 x PBS = 8 : 1 : 1
(*)hepes buffer
 Hepes100 mM
 MgSO42 mM
Add NaOH to pH6.9 and autoclave

Hybridization solution
deionized formamide50%
SSC (pH7, autoclaved)5x
sonicated salmon testis DNA100 μg/ml
yeast tRNA100 μg/ml
heparin100 μg/ml
Tween 200.1%

CHAPS (DOJINDO 349-04722)

IMMUNO pen (Wako, Japan)

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