This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
CHAPTER 9 - MOLECULAR METHODS
Whole-Mount in situ Hybridization
(Source: S. Schulte-Merker)
1. Fix embryos with 4% paraformaldehyde in
PBS overnight at 4°C.
2. Wash twice in PBS, 5 min each, at
3. Remove the embryos from their chorions
using watchmaker forceps (easiest at this
4. Transfer embryos to vials with 100%
methanol (MeOH), replace with fresh
methanol after 5 min. Use MeOH and not
ethanol, since ethanol causes a higher
Note: From this point on, the embryos remain in the same glass
vial until they are ready for prehybridization.
the embryos to -20°C for
at least 30 min
(embryos can be
stored that way for
months; this step is
embryos even if you
don't want to store
6. Bring the embryos back to
rse 5 min in 50%
MeOH in PBST and
then 5 min in 30%
8. Rinse twice in PBST for 5 min
Note: Embryos can also be removed from their chorions at this
point, but chorions are sticky after having been in
9. Fix for 20 min in 4% paraformaldehyde in
PBS at RT.
twice in PBST for 5
Proteinase digestion and postfixation
1. Digest with proteinase K (10 µg/ml in
PBST) at RT for 5 to 12 min (depends on
the stage; younger stages are more sensitive;
depends, also, on the batch of enzyme; we
test each new batch).
2. Rinse briefly in PBST; wash for 5 min in
3. Fix as above (4% paraformaldehyde in PBS,
as above (2 times in
Acetic anhydride treatment (optional)
1. Replace PBST with dH2O (as
2. As quickly as possible, replace H2O with a fresh mixture of 2.5
µl acetic anhydride in 1 ml of 0.1M
3. Incubate for 10-60 min at
4. Wash 2 times for 10 min each in
This treatment helps reduce background from endogenous phosphatases
and is therefore only necessary in cases where background is a problem. It is
also unnecessary if peroxidase-coupled anti-digoxigenin antibodies are used.
1. Transfer embryos (up to 40) into small
eppendorf tubes (0.8ml) in approximately
300 µl of HYB*.
2. Incubate 5 min at 55°C;
afterwards, replace HYB*
with an equal volume of HYB+.
3. Prehybridize at 55°C for
1-48 hr in HYB+.
Prepare RNA Probes according to the Boehringer instructions (Cat.
#1175025). About 5 to 10 µg of digoxigenin-labeled probe is transcribed
from 1 µg of a linearized plasmid. Hydrolyze the probes to an average
length of 150-300 nucleotides following the protocol of Cox et al.
(1984; Devel. Biol.101:485-502). After the final
precipitation, the hydrolyzed probe should be taken up directly in HYB+ and
stored at 20°C.
1. Remove as much of the preHYB+ as possible without letting the embryos touch air.
2. Add 20 to 40 µl of fresh HYB+ containing 20-100 ng of RNA
probe (about 0.5-5.0 ng/µl) so that all
embryos are covered by the solution. Heat
the probe in HYB+ for 5
min at 68°C before
adding to the embryos. For some probes,
signal intensity decreases below 10 ng;
amounts higher than 100 ng don't usually
give a higher signal intensity. Probably the
amount of probe to add depends upon the
amount of RNA you want to detect and has
to be titrated.
3. Incubate overnight at 55°C.
Remove probe (probes can be reused twice; using them more often results
in weaker signals. The probes in HYB+ are stable for at least
half a year).
embryos for 20 min at
50% formamide in
2. Rinse 3 times for 10 min each at 37°C in
3. Rinse for 5 min at 37°C
4. Digest for 30 min at 37°C
in RNAse (RNAse A, 20 µl/ml plus
RNAse T1, 100 U/ml in
5. Rinse 10 min at 37°C in
6. Soak 60 min at 55°C in
50% formamide in
7. Rinse 15 min at 55°C in
8. Rinse twice for 15 min each at 50°C in
9. Rinse 5 min in PBST at
10 Transfer embryos to microliter-
1. Soak embryos 2 times for 30 min each at
55°C in 50% formamide
2. Rinse 15 min at 55°C in
3. Rinse 2 times for 30 min each at 55*C in
4. Transfer embryos to microliter-
IMPORTANT: For some probes, the RNAse treatment is
unnecessary and decreases the signal intensity. For other probes, omitting the
RNAse leads to an unacceptably high background. It is therefore advisable to test
whether the RNAse treatment is necessary for any given probe. If you can do
without it, follow Option B; if not, use Option A.
1. Block for 1 hour at RT with PBST plus
blocking reagent (skimmed milk, new born
calf serum, BSA, etc.; the Fab-fragments
are not very sticky, so it doesn't seem to
matter what one
Fab-AP as supplied
by Boehringer at a
and shake for 4 hours
at RT in PBST plus
4 times for 25 min
each with PBST plus
4. Wash 3 times for 5 min each in staining
ate in staining buffer
with 4.5 µl
NBT and 3.5 µl
75 mg/ml in 70%
6. Stain for 30 min to
7. Wash in PBS.
8. Dehydrate with 100% MeOH twice (10 min
each) and mount in a 2:1 mixture of
benzylbenzoate:benzylalcohol. This mixture
has the same refractive index as yolk and
clears the embryos very
If one uses alkaline phosphatase as a detection enzyme one has to be aware
that NBT/X-Phosphate will fade in anhydrous solutions. Fixing the embryos after
the color reaction is necessary if you want to clear the embryos in alcohol. After
fixation (4% paraformaldehyde at RT for at least half an hour) even weaker
signals are reasonably stable in alcohol. You can also overstain with careful
monitoring of the dehydration and clearing process. Another alternative is to
photograph weak signals immediately after transferring the specimen to alcohol
or to clear them in glycerol.
In situ hybridization
PBST - PBS plus 0.1% Tween
SSCT - SSC plus 0.1% Tween
HYB* - 50% formamide, 5xSSC, 0.1% Tween-20
HYB+ - HYB* with 5mg/ml torula (yeast) RNA, 50µg/ml heparin
The torula RNA is prepared by proteinase K digestion of RNA with
subsequent phenol-, phenol-chloroform-, and chloroform-extraction. The RNA
is precipitated and dissolved in DEPC-treated water. HYB* and
HYB+ should be kept at -20°C
In situ Hybridization staining buffer
100 mM Tris pH 9.5
50 mM MgCl2
100 mM NaCl
1 mM Levamisol (add fresh)
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