1) Collect the embryos (anywhere between 0-15 hrs) and rinse thoroughly with water.
2) Dechorionate in 50% "Clorox" (commercial bleach) for 2-5 min.
3) Wash with water.
4) Transfer the embryos (up to 3ml volume of embryos) into a polypropylene tube
containing:
16 ml 0.1 M Hepes, pH 6.9
2 mM Mg-sulfate
1 mM EGTA
4 ml 20% paraformaldehyde (dissolve
paraformaldehyde in boiling water.
You may have to add NaOH to get it into
solution)
20 ml heptane
5) Place on a rotator for 15 to 20 min.
6) Remove the lower phase (fixative) and add 15 ml methanol and shake the tube
vigorously for 10-30 sec. The devitellinized embryos will sink to the bottom (heptane
will be on top and the vitelline membranes will stay at the interface).
7) Transfer embryos together with some methanol into a new tube. Wash with
MeOH:EGTA (90% methanol;10% 0.5 M EGTA, pH 8.0).
8) Prepare 4% paraformaldehyde in PBS (PP; made by diluting 20% paraformaldehyde
solution with 1X PBS) and MeOH:EGTA (ME) solution (see above). Rehydrate the
embryos as follows:
5 min in 7 parts ME / 3 parts PP
5 min in 5 parts ME / 5 parts PP
5 min in 3 parts ME / 7 parts PP
20 min in PP
9) If you want to store the embryos at this point, wash them in PBS and dehydrate
them through 30%, 50% and 70% ethanol and store at -20°C; rehydrate before
pretreatment.
II. Pretreatment:
All the following incubation steps are performed at room temperature. Avoid
potential RNAase contaminations. The PBS can be treated with DEPC and autoclaved
and then supplemented with 0.1% Tween 20 (=PTw). We have found that the DEPC
treatment is not absolutely necessary.
1) Wash the embryos 3 times for 5 min each in PTw
2) Incubate the embryos for 7-8 min in PTw plus 50 µg/ml non-predigested Proteinase
K (Boehringer Cat# 745 723; you may have to determine the optimal extent of
treatment for your batch of proteinase K; the optimal time seems to be 2-3 min before
the embryos begin to show any noticable damage).
3) Stop Proteinase K digestion; by incubating for 2 min in 2 mg/ml glycine in PTw.
According to Tautz andf Pfeifle, the Proteinase K digestion is a critical step. If it is too short, you lose sensitivity and increase the background. If it is too long, the embryos tend to disintegrate during hybridization.
4) Wash 2 times for 5 min each with PTw.
5) Refix for 20 min with PP.
6) Wash 5 times for 5 min each in PTw.
Preparation of DNA Probe
I. PCR-labeled Single-stranded Probe ;from Cloned DNA:
Using this PCR technique, single stranded DNA probes containing digoxigenin
can be made for in situ hybridization. The biggest advantage of this technique over the
random oligo priming method is that a much larger quantity of probe can be made with
the same amount of starting nucleotide. In addition, the ratio of labelled DNA to
unlabelled starting material (which will compete for hybridization) is much higher. In
situations where transcripts are not very abundant, these single-stranded probes seem to
work better than the oligo primed probes. The major disadvantage is that it is difficult
to control the probe size. The PCR reaction will generate full length strands for inserts
up to 2-3 kb. We simply boil the DNA to reduce its size, but have not made any
estimates of how effective this really is nor have we investigated other alternatives to
this problem. Empirically, however, this labelling technique works extremely well for the
in situs. In addition, we have found that biotin-16-dUTP; can also be incorporated this
way and used for in situs using streptavidin-alkaline phosphatase for the detection
step. This method works okay, but is clearly 2 to 3- fold less sensitive than the
digoxigenin. Also, PCR can be used to generate very large amounts of biotin-labelled
double-stranded DNA by the use of two primers as long as the distance between
primers is less than 2-3 kb. Finally, we have observed that biotin-labelled DNA runs
normally in agarose gels, but digoxigenin-labelled DNA; will run as though it is larger
than it actually is.
1) Prepare the following stock solutions:
10 x React 0.5 M KCl
(comes in PCR kit) 0.1 M Tris-HCl, pH 8.3
15 mM MgCl2
0.01% (w/v) gelatin
5 x dNTP Mix 1 mM dATP 7.1 µl of 10mM
(equivalent to 1 mM dCTP 7.1 µl of 10mM
tube 6 of Genius 1 mM dGTP 7.1 µl of 10mM
kit) 0.65 mM dTTP 4.6 µl of 10mM
0.35 mM Digoxigenin-11-dUTP; 25.0 µl of 1mM
(Dig-dUTP can be purchased 20.5 µl of H2O
as a 1mM soln from Boerhinger) 71.4 µl total vol.
SK primer 30 ng/µl (approx 5.3 µM)
KS primer 30 ng/µl (approx 5.3 µM)
Linearized DNA containing insert. Linearize Bluescript DNA as you would to make
an RNA runoff probe. For example, you might have a 2.0 kb EcoR1 fragment cloned
into the R1 site of Bluescript. You might linearize by cutting one aliquot with Kpn and
another with SacII (assuming there are no Kpn or SacII sites in the insert). The Kpn
cut DNA can be used with the SK primer to create one strand which might be the
anti-sense strand. The SacII cut DNA can be used with the KS primer to create the
sense strand which can be used as a control in the hybridization. T3, T7, M13, or
internal primers can be used in place of the KS/SK primers. We have on occasion cut
DNA and then not bothered to phenol extract and precipitate afterwards, but have
simply heat inactivated the restriction enzyme and then successfully used it for the
PCR reaction. You will want to dilute your DNA with water to a final concentration
of about 100-200 ng/µl. Keep in mind that if your insert is much over 3 kb that the
probe produced will probably not be fully representative of the entire insert .
2) Set up the following reaction (25 µl volume):
water 8.5 µl
10 x React 2.5 µl
5 x dNTP mix 5.0 µl
KS (or SK) primer 5.0 µl
DNA (100-200 ng/µl) 2.0 µl
Add 40 µl mineral oil and centrifuge. Boil 5 min then add Taq:
Taq I DNA Polymerase 2.0 µl (1.25 units total; a 1:8 dilution in
25.0 µl water of 5U/µl Taq stock)
3) Mix the contents and then centrifuge for 2 min. Incubate for 30 cycles in the PCR
thermal cycler under the following conditions:
95°C for 45 seconds
55°C for 30 seconds
72°C for 1 minute
4) After the PCR run, add 75 µl dH20, then centrifuge.
5) Remove 90 - 95 µl of the reaction from beneath the oil.
6) Do two ethanol precipitations as follows. Add NaCl to 0.1 M and 3 vols of 100%
EtOH. You can use 10 µg of glycogen or tRNA as a carrier (0.5 µl of a 20 mg/ml
stock). Mix well, -70oC for 30 min, and centrifuge. Wash with 70% ethanol.
Speedvac dry. 2nd precipitation is optional.
7) Resuspend pellet in 300µl of hyb buffer (hyb. buffer of Tautz and Pfeifle protocol below). For efficient penetration and hybridization to the embryos, the average probe length should be about 50-200 bp. To reduce the size of the single-stranded DNA, boil the probe for 40-60 min. The probe can be diluted as much as ten-fold before use, but this varies quite a bit depending on the abundance of your transcript and how much of a problem you have with background staining. I recommend that you use the probe anywhere from undiluted (original 300µl) to three-fold diluted for your first attempt.
8) Check of probe (solutions are same as those described in the embryo protocol below):
1. Remove 1 µl of probe. Add 5 µl of 5X SSC. Boil 5 min. Quick cool on
ice.Centrifuge.
2. Spot 1-2µl onto a small nitrocellulose strip that will fit into an eppendorf tube
or 5 ml snap cap tube.
3. Bake between two sheets of Whatman paper in an 80°C vacuum oven for 30
min. The residual formamide may cause the nitrocellulose to warp. If this is a
problem, reduce the time in the baking oven or do this spot test before the
second precipitation. Note: unincorporated nucleotide binds only slightly to
the nitrocellulose.
4. Wet the filter with 2X SSC, wash 2X 5 min in PBT. Place strip into eppendorf
tube or 5 ml snap cap tube.
5. Block 30 min in PBT. Incubate in PBT + antibody (1:2000) for 30-60 min.
6. Wash 4 x 15 min in PBT.
7. Wash 2 x 5 min in NaCl/MgCl2/Tris/Tween solution (Levamisole is not
needed).
8. Develop with NBT and X-phosphate as described for embryos. Spots should
be visible within a few minutes and dark by 10-15 min.
II. Preparation of probe by oligolabelling
An alternative to PCR-generated probes is to create probes by the random
oligo-priming technique. This is the modification by Charlie Oh (Kornberg lab) of the
protocol that is described in the Genius Kit. By increasing the random primer
concentration and lowering the temperature, smaller probe fragments are created
resulting in a higher signal and less background.
10X LB 475 µl 1M Pipes pH 6.6
25 µl 1M MgCl2
3.3 µl ßME
Labelling reaction:
1) Prepare 200ng of purified insert DNA in 9 µl of water.
2) Boil 10 min.
3) Quick freeze in dry ice/ethanol or liquid nitrogen.
4) Add following components on ice.
10X LB 2.0 µl
hexamer 6.0 µl of a 10 mg/ml soln. (Random hexamers;
Pharmacia # 27-2166-01)
dNTP 2.0 µl (tube 6 of Genius Kit)
Klenow 1.0 µl (5 Units)
5) Incubate at 15°C for 1 hr.
6) Transfer to room temp for 3 hrs.
7) Precipitate probe and resuspend in 200µl of hyb buffer.
Hybridization and detection:
1) Hyb solution: 50% formamide
5x SSC
100 µg/ml sonicated salmon sperm DNA
50 µg/ml heparin
0.1% Tween 20
2) Wash embryos 10 min in a 1:1 mix of hyb soln. and PTw.
3) Wash embryos 10 min in hyb soln.
4) Prehybridize in heat denatured hyb soln for about 1 h at 45°C. We have found that
embryos can be stored in the hyb soln at -20°C for 1-2 weeks.
5) Boil the probe for 10 min and then cool on ice. Remove most of the prehyb soln from the embryos and add 100-200µl of probe. About a 30µl volume of embryos in the 100-200 µl of probe works well.
6) Mix and hybridize overnight in a 45°C waterbath. Round bottom Nunc tubes work well for this. After the hybridization, the probe can be recovered and used repeatedly. We have been able to use the same bit of probe up to six times. The repeated boiling may improve the signal by further reducing the size of the probe.
7) Wash embryos 20 min in Hyb soln. All washes in steps 7 and 8 should be done with
agitation at 45oC. You will want to pre-mix and pre-warm all the solutions.
8) Wash embryos 20 min each in: 4 parts Hyb. soln. / 1 part PTw
3 parts Hyb. soln. / 2 parts PTw
2 parts Hyb. soln. / 3 parts PTw
1 part Hyb. soln. / 4 parts PTw
2 times 20 min in PTw
9) Wash embryos 2 times 20 min each with PBT. This and all subsequent steps are
done at room temp.
PBT = 1X PBS, 0.1% BSA, 0.2% Triton X-100.
10) Dilute the alkaline phosphatase conjugated goat anti-digoxigenin antibody (vial 8 of
the Genius Kit) to 1:2000 - 1:2500 with PBT. Incubate the embryos for 1 hour with
200-400µl of the dilute antibody soln.
11) Wash 4 times 20 min each in PBT.
12) Wash 3 times 5 min each in 100 mM NaCl
50 mM MgCl2
100 mM Tris, pH 9.5
0.1% Tween 20
1 mM Levamisol (Sigma #L-9756; add fresh
just before use. Levamisole is
an inhibitor of potential endogenous
phosphatase. Nipam has
done experiments where it has been left
out and there does not seem to be any
background problem.)
Prepare a solution of NaCl/MgCl2/Tris/Tween/Levamisole containing 4.5 µl of NBT (vial 9 of Genius kit) and 3.5 µl of X-phosphate (vial 10) per ml of solution. Add 0.5-1.0 ml to the embryos after the last wash. You will see the signal appear anywhere between 10-60 min. Stop by washing several times in PBS. We have tried alternative substrates. Vector Kit II substrate produces a brown/black product that is insoluble in xylene and can therefore be mounted in permount. This substrate diffuses less and allows for the resolution of sub-cellular localization, but it is 2-3 fold less sensitive than the standard method. We have not had any luck using an HRP enzyme detection system.
13) The embryos reacted with NBT/X-phosphate should be transfered to 50% glycerol
in PBS for 1-2 hrs and then transfered to 70% glycerol in PBS. They can be stored at
4°C for several weeks.
Some Final Notes:
If the embryos are prepared properly, the morphology can be excellent. The
embryos can be dissected in the 70% glycerol soln if needed. One additional possibility
is to reduce the level of background by digesting with a single-stranded nuclease after
the hybridization with a single-stranded probe. This would be equivalent to the RNAse
A digestion of 35S RNA in situs. This should destroy the unbound probe and leave the
RNA/DNA hybrids intact. It may be tricky as the single-stranded nucleases can be
difficult to control. The protocols described above are based on experiments performed
by me, Nipam Patel, Yash Hiromi and Elinor Fanning using a variety of different gene
probes.
REAGENTS:
Genius Kit. Boehringer Cat# 1093 657. Alternatively, the components can be purchased
individually. Several are listed below.
Digoxigenin-11-dUTP Boehringer Cat# 1093-088, 25 nmol/25 µl (1mM)
Anti-Digoxigenin-AP Conjugate Boehringer Cat# 1093-274
NBT is 4-Nitro blue tetrazolium chloride, Boehringer, Cat# 1087-479
X-phosphate is 5-Bromo-4-chloro-3-indolyl-phosphate, Boehringer, Cat# 760-994