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Anderson Lab In Situ Hybridization Protocols
March 1997
I: In Situ Hybridization of Frozen Sections
Sections are collected on Superfrost/Plus slides purchased from Fisher and dried
in air for one hour at room temperature, then stored at 20°C. Alternatively, use RNAse
free slides coated with TESTA, in which case you must dry two hours. It is sometimes
necessary to wash the slides in DEPCPBS and three changes of DEPCwater before
storing at 20°C. This depends on both the probe and embedding material used. Details
of how to prepare TESTA coated slides are in the Appendix.
A: PreTreatment of Sections
N.B. All reagents and mailers in the following steps must be RNAsefree.
In this section, steps 6 and 7 (the acetylation) is vital for some probes, but raises the
background for others. Some probes, like SCG10, Brn3 and neurofilament, work
best without acetylation. Other probes, like GATA2, require it. Some work okay
without it but have improved signal to background if acetylation is included. I
recommend comparing sister slides with and without acetylation the first time you
try a probe.
1. Warm slides to room temperature and dry at 50°C for 15 minutes.
2. Fix in 4% paraformaldehyde in DEPCPBS at room temperature for 20 minutes.
3. Wash twice in DEPCPBS at room temperature for 5 minutes.
4. Treat slides with 50µg/ml Proteinase K in PK buffer at room temperature for between
815 minutes depending on the age of the embryo.
5. Wash once in DEPCPBS at room temperature for five minutes. Fix in 4%
paraformaldehyde in DEPCPBS for 15 minutes.
6. Rinse once in DEPCwater.
7. Place slides in an RNAsefree glass trough with a stir bar. Add 250ml 0.1M RNAse
free triethanolamineHCl pH 8.0. Add 0.625ml acetic anhydride (CARE!) with constant
stirring. Turn off stirrer when the acetic anhydride is dispersed and leave for a further 10
minutes.
8. Wash slides in DEPCPBS at room temperature for five minutes.
9. Prehybridise for 34 hours at 60°C. Replace with 12µg/ml of probe and continue
incubation for a further 1216 hours. These steps should be performed in the
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hybridization oven, not the regular 60°C oven, as the latter does not maintain its
temperature well.
N.B. We have found that the most effective way to carry out the hybridisation is in slide
mailers. It is a good idea to thoroughly seal the lids of the slide mailers with parafilm to
prevent evaporation of probe.
B: Washing Steps
N.B. After hybridisation, it is not necessary to use RNAsefree buffers.
1. Place slides in a trough with a stir bar. Wash in 1xSSC at 60°C for 10 minutes.
2. Wash in 1.5xSSC at 60°C for 10 minutes. Cool slides to 37°C.
3. Wash twice in 2xSSC at 37°C for twenty minutes each.
4. Treat with 0.1µg/ml RNAse A in 2xSSC at 37°C for 30 minutes.
5. Wash in 2xSSC at room temperature for 10 minutes.
6. Wash twice in 0.2xSSC at 60°C for 30 minutes each.
7. Wash once in 0.2xSSC at room temperature for 15 minutes.
8. Wash once in PBT for 15 minutes.
9. Incubate slides in 20% heatinactivated sheep serum in PBT for between one and five
hours at room temperature. For some probes, the longer incubation seems to cut down
on background.
C: Antibody Visualisation of Digoxygenin
1. Incubate slides with preabsorbed antidigoxygenin antibody (coupled to alkaline
phosphatase) diluted to a final concentration of 1:2000 in 20% sheep serum in PBT at
4°C overnight.
2. Wash three times in PBT at room temperature for 30 minutes each.
3. Wash twice in Alkaline Phosphatase buffer at room temperature for 5 minutes each.
Levamisole is sometimes included in the second wash and reaction buffer as an
inhibitor of endogenous alkaline phosphatase, but on most embryo sections the
endogenous activity does not survive the hybridization procedure.
4. For every ml of Alkaline Phosphatase buffer, add 1µl of NBT and 3.5µl of BCIP, and
develop in the dark for between 220 hours, depending on the abundance of the RNA.
Since the alkaline phosphatase enzyme is very stable, it is possible to wash out the
NBT/BCIP, replace with alkaline phosphatase buffer , and to continue the reaction at a
later time.
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5. Wash twice in PBS to remove substrates.
6. Fix slides in MEMFA for at least 15 minutes at room temperature. Mount slides in
glycerol/PBS.
N.B. If the BCIP/NBT precipitate is not fixed, it will slowly darken over the course of
about a month. Eventually, the background will become too dark.
Additional Protocols for sections: Double label protocols.
These modifications were contributed by Tetsuchiro Saito.
Modifications for two probe double label protocol
This protocol works best for demonstrating two populations of cells expressing two
separate markers, since both products are cytoplasmic, and the NBT/BCIP product
(purple) can obscure the INT/BCIP product (red).
A: Preparation of probes.
1. A probe is prepared by using fluorescein RNA labeling mix in place of digoxygenin
NTPs.
B: Hybridization.
Both the digoxygenin labeled probe and the fluorescein labeled probe are included in
the hybridization buffer at 12µg/ml each.
C: Staining.
The following steps are performed in slide mailers.
1. Sections are incubated with either antifluoresceinAP Fab fragments or anti
digoxygeninAP Fab, preabsorbed and at the appropriate dilution in 20% sheep serum
in PBT, at 4°C overnight. Stain the weaker probe first.
2. After washing, stain with NBT/BCIP as in the normal protocol, until staining reachs
the desired intensity.
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3. Wash three times in PBS at room temperature for 5 minutes each to remove substrates.
4. Incubate in TE (100mM TrisHCl pH 7.5, 50mM EDTA) at 85°C for 10 minutes to
inactivate alkphos. Use the 68°C water bath turned up to 85°C.
5. Wash three times in PBS at room temperature for 5 minutes each.
6. Wash once in PBT at room temperature for 10 minutes.
7. Incubate slides in 20% heatinactivated sheep serum in PBT at room temperature for
30 minutes.
8. Incubate slides with the other antibody at 4°C overnight. If you used antifluorescein
originally, use antidigoxygenin as the other antibody, and vice versa.
9. Wash three times in PBT at room temperature for 30 minutes each.
10. Wash twice in Alkaline Phosphatase buffer at room temperature for 5 minutes each.
11. For every ml of Alkaline Phosphatase buffer, add 7.5µl INT/BCIP stock solution, and
develop in the dark for between 220 hours, depending on the abundance of the RNA.
N.B. AntidigoxygeninAP Fab final concentration is 1:2000. AntifluoresceinAP is used
at 1:4000.
Modifications for one probe, one antibody double label protocol
Since each antibody epitope can vary in stability, this protocol must be specifically
tailored for the antibody used. Tetsuchiro does not recommend trying membrane or
cytoplasmic antigens for double label; nuclear antigens best survive the hybridization
procedure. This is the protocol I have used for combining the quail nuclear antigen with
in situ hybridization. (PMW)
A: PreTreatment of Sections
Test different proteinase K concentrations to find which provides the best compromise
between in situ signal and antibody signal. For example,
100% proteinase K 50µg/mL
10% proteinase K 5µg/mL
1% proteinase K 0.5µg/mL
substituted into the normal hybridization procedure. Also try the following:
1. Warm slides to room temperature and dry at 50°C for 15 minutes.
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2. Fix in 4% paraformaldehyde in DEPCPBS at room temperature for 20 minutes.
3. Wash twice in DEPCPBS at room temperature for 5 minutes.
4. Prehybridise for 34 hours at 60°C. Replace with 12µg/ml of probe and continue
incubation for a further 1216 hours.
B: Washing Steps
Follow the normal protocol for stringency washes through the 15 minute wash with PBT
(steps 1 8).
9. Block with 1% goat serum, 1% BSA, 0.5% Triton in tissueculture grade DPBS at room
temperature for 1 hour.
10. Incubate overnight with 1:1 QCPN supernatant and 1% goat serum, 1% BSA, 0.2%
Triton in TC DPBS.
C: HRP Development
1. Wash three times in PBT at room temperature for 5 minutes each.
2. Wash three times in PBS at room temperature for 5 minutes each.
3. Inactivate the endogenous peroxidase with 0.3% H2O2 in methanol at room
temperature for 30 minutes. I have tested slides pretreated with 1µg/mL proteinase K
and hybridized at 60°C for 18 hours, followed by normal SSC washes, and have found
residual endogenous peroxidase activity in the red blood cells.
4. Wash three times in PBS at room temperature for 5 minutes each.
5. Wash three times in PBT at room temperature for 5 minutes each.
6. Incubate with secondary antibody (goat anti mouseHRP, in this case) in 1% goat
serum, 1% BSA, 0.1% Triton in TC DPBS.
7. Wash three times in PBT at room temperature for 5 minutes each.
8. Wash once in PBT at 4°C overnight to further reduce background, if necessary.
9. Wash three times in acetate imidazole buffer at room temperature for 5 minutes each.
10. Develop HRP reaction with nickelDAB in acetate imidazole buffer.
11. Wash three times in PBS at room temperature for 5 minutes each.
12. Wash once in PBT at room temperature for 10 minutes.
13. Incubate slides in 20% heatinactivated sheep serum in PBT at room temperature for
one hour.
Now follow the normal protocol for visuallization of digoxygenin (or fluorescein.)
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Very important: Fixing the slides in MEMFA eliminates the NiDAB signal! Mount and
view the slides right away.
Troubleshooting and common problems.
A. Low signal / high background
Is your oven maintaining temperature? Are people going in and out of it? If you’re
using a hybridization oven, you can also consider trying 68°C or 72°C.
If you are reusing the 20% sheep serum block or antidigoxygenin antibody, check it for
particulates, or just replace it. (especially for spotty background)
Be extremely rigorous about how long your slides are in 0.2X SSC. This highstringency
step strips the probe off the sections. It is necessary to reduce background, but I have
found that with clean probes, the highstringency washes can be pared back to 25
minutes and this improves signal. The same is true for the RNAse A step. (PMW)
Did you hydrolyze your probe? Poor signal is also common if the probe is less than 500
bp. (Hai rarely hydrolyzes his probes, though, and his wholemounts are lovely.)
Check for particulates in the phosphate buffer used in making the fixative or sucrose for
your animals. Contaminated PB used to make either reagent causes unholy background
for antibody staining, and severely reduces signal for in situ.
B. Uneven labeling across the short axis of the slide.
There is some debate as to what causes this phenomenon. Andy says to reduce the
speed of the stir bar during the SSC washes to under 3. Pat says it’s evaporation of
formamide during hybridization, and to use fresh probe next time. Try both.
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II: Whole Mount In Situ Hybridization.
Embryos should be dissected free of any extraembryonic membranes, fixed in
4% paraformaldehyde in DEPCPBS for 2 hours at room temperature (or 4°C
overnight), washed in DEPCPBS, and then stored in 100% methanol at 20°C until
required. Hai punctures the embryos on the forehead and hindbrain region with a fine
needle to allow free exchange of reagents.
In general, we do the washes and incubations in 15 ml tubes containing about 56
ml liquid. The tubes are rocked gently on a rotating platform to allow thorough
exchange of solutions.
A: PreTreatment of Embryos
1. Bleach the embryos in methanol/peroxide (5 volumes methanol, 1 volume 30% H2O2)
at room temperature for three to five hours.
2. Wash twice in 100% methanol 5 minutes at room temperature.
3. Rehydrate the embryos in:
75% MeOH : 25% PTw 5 minutes at room temperature
50% MeOH : 50% PTw " " "
25% MeOH : 75% PTw " " "
100% PTw " " "
Wash twice in PTw for 5 minutes each.
4. Treat embryos with 10µg/ml proteinase K in PTw for 560 minutes. This is a critical
step, as overdigestion will destroy the embryos, and underdigestion will give a poor
signal. Exact times should be determined for each embryo species and age; for example,
10 minutes is fine for E9.5 mouse. Treat the embryos very gently after this step until
they are refixed.
5. Rinse twice gently in PTw. Refix embryos in 4% paraformaldehyde for 30 minutes at
room temperature.
6. Wash twice in PTw for five minutes each at room temperature.
7. Transfer embryos to a 2ml Eppendorf tube. Remove as much liquid as possible, taking
care to avoid damaging the embryos. It's better to remove too little than too much.
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Replace with 1 ml hybridisation mix. Remove this mixture after the embryos sink, and
replace with fresh hybridisation mix.
8. Prehybridise the embryos at 63 70 °C for between 1 and 4 hours. Hai uses a heat
block. Add probe to a final concentration of about 1µg/ml, and hybridise overnight at
the same temperature.
(N.B. The subsequent washes with hybridisation mix can be quite costly. The Appendix
contains an alternative hyb recipe that uses much less tRNA and works fine for whole
mounts. I've tried it a couple of times on sections and it seems to work OK too, but
proceed with caution.)
B: Washing Steps
1. Wash three times with prewarmed (70°C) hybridisation mix for five minutes each in
the same tube.
2. Wash twice with prewarmed hybridisation mix for 30 minutes each at 70°C.
3. Wash once with a 1:1 mixture of hybridisation mix and TBST (prewarmed) at 70°C
for 20 minutes.
4. Wash twice with TBST at room temperature for five minutes each.
5 Incubate embryos in 50 µg/mL RNaseA in TBST at 37°C for one hour with gentle
rocking.
6. Wash three times with TBST for five minutes each at room temperature.
7. Wash twice in formamide wash solution for 30 minutes each at 60°C
8. Wash three times with TBST for five minutes each at room temperature.
9. Block embryos with TBST containing 10% sheep serum for 1 3 hours at room
temperature.
C: Antibody Visualisation of Digoxygenin
1. Incubate with preabsorbed antidigoxygenin antibody diluted to a final
concentration of 1:2000 at 4°C overnight with gentle rocking.
2. Wash three times with TBST with 2mM levamisol for five minutes each at room
temperature.
3. Wash five times with TBST at room temperature for one hour each.
4. Wash overnight with TBST at 4°C. This sounds excessive, but it helps clean up the
background.
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5. Wash twice with Alkaline Phosphatase buffer at room temperature for 30 minutes
each.
6. For every ml of Alkaline Phosphatase buffer, add 1µl of NBT and 3.5µl of BCIP, and
develop in the dark for between 220 hours, depending on the abundance of the RNA.
The product should usually be visible in an hour or two.
7. When the reaction has proceeded to your satisfaction, it is imperative to quickly stop
the reaction to prevent excess background. Wash three times in TBST for five minutes
each in the dark.
8. Fix in 4% paraformaldehyde at room temperature for 30 minutes or overnight at 4°C
in the dark.
9. Dehydrate with methanol and TBST series for five minutes each at room temperature:
25% methanol and 75%TBST
50% : 50%
75% : 25%
10. Incubate in 100% methanol for 10 minutes at room temperature. This darkens the
reaction product from purple to a deep blue.
11. Rehydrate to TBST using the series in reverse.
12. Wash twice in TBST for five minutes at room temperature
13. Clear the embryos in 4 : 1 glycerol:water for photography.
14 If sectioning is desired, do not immerse in glycerol. Sink in 15% sucrose and freeze in
OCT, or embed in 8% gelatin/15% sucrose.
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III: In Situ Hybridization on Cultured Cells
This protocol is typically used for cells or explants cultured in 35mm dishes, but
can be adapted to coverslips. In general, the signal tends to come up much more slowly
than in either sections or whole mounts.
A: PreTreatment of Cells
1. Fix in 4% paraformaldehyde in DEPCPBS at room temperature for 10 minutes.
2. Wash three times in DEPCPTw at room temperature for 5 minutes each.
3. Permeablilize with 0.2 M HCl for 10 minutes at room temperature.
4. Wash two times in DEPCPTw for 5 minutes each.
5. Digest with 10 ug/ml of Proteinase K in DEPCPTw for 1030 minutes at room
temperature. Ten minutes is typically sufficient, but you may wish to vary this
incubation depending on the probe and the nature of the cultured tissue. Longer
digestions may improve the signal but overdigestion causes cells to fall off the plate
during the hybridization and washing steps.
6. Rinse once very carefully with DEPCPTw.
7. Fix again in 4% paraformaldehyde in DEPCPBS for 15 minutes.
8. Wash three times in DEPCPTw for 5 minutes each.
9. To 25ml 0.1M triethanolamine, pH8.0, add 62.5µl acetic anhydride and quickly mix
until thoroughly dispersed. Incubate cultures in this mixture for 10 minutes at room
temperature.
10. Wash cultures in 1x DEPCSSC for five minutes at room temperature.
11. Prehybridize for 6 hours at room temperature or 3 hours at hybridization
temperature. Remove prehyb and add probe at a final concentration of between 1 and
2µg/ml. Hybridize overnight at 60°C.
N.B. To prevent evaporation, incubate in a tightsealing tupperware box containing
towels soaked in 50% formamide and 5xSSC.
B: Washing Steps
1. Wash once in 1x SSC at hybridization temperature for 10 minutes.
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2. Wash once in 1.5x SSC at hybridization temperature for 10 minutes. Then cool to
approximately 37 C.
3. Wash twice in 2x SSC at 37 C for 20 minutes each.
4. Treat with 0.2 ug/ml of RNAse A in 2x SSC at 37 C for 10 30 minutes. Ten minutes
is typically sufficient, though longer RNAse treatment may lower the background
and/or signal depending on the probe.
5. Wash once in 2x SSC for 10 minutes at room temperature.
6. Wash twice in 0.2x SSC at hybridization temperature for 30 minutes each.
7. Wash twice in PTw at hybridizaton temperature for 10 minutes each.
8. Wash once in PTw for 10 minutes at room temperature.
9. Wash once in PBT for 15 minutes at room temperature.
10. Incubate in 20% sheep serum in PBT for 3 hours at room temperature.
C: Antibody Visualisation of Digoxygenin
1. Incubate cultures with antidigoxygenin antibody (coupled to alkaline phosphatase)
diluted to a final concentration of 1:1000 in 20% sheep serum in PBT at 4°C overnight, or
for two hours at room temperature. It is not necessary to use preabsorbed antibody,
although it doesn't hurt.
2. Rinse three times in PBT.
3. Wash four times with PBT at room temperature for 10 minutes each.
4. Wash twice in Alkaline Phosphatase buffer (first wash without levamisole, second
wash with) at room temperature for 10 minutes each.
5. For every ml of Alkaline Phosphatase buffer, add 4.5µl of NBT and 3.5µl of BCIP, and
develop in the dark for between 236 hours, depending on the abundance of the RNA. It
may be necessary to wash the cultures and add fresh reaction mixture after 12 hours or
so.
6. When the reaction has proceeded far enough, wash in PBT, and fix in MEMFA.
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Appendix: Additional Techniques
A: Preparation of RNAse Free Slides
1. Soak VWR slides overnight in Dichrol at room temperature in a fume hood. Wash the
slides thoroughly to remove any residual Dichrol, rinse for one hour in running water,
then for a further hour in running distilled water.
2. Dry slides at 150°C for 20 minutes.
3. Dip slides in a 2% solution of TESTA (3aminopropyltriethoxysilane; Sigma A3648) in
dry acetone for 5 minutes.
4. Wash in 2 changes of acetone and three changes of DEPCwater. Dry overnight at
42°C and store dry. TESTA slides should be used within 6 weeks, as their adhesive
properties tend to fade after this time, i.e. your sections will fall off during
hybridization, and you will have to do it all over.
B: Probe Preparation
1. Cut between 20 and 40µg of maxiprep quality plasmid DNA with a fivefold excess
of an appropriate restriction enzyme for 2 hours. Check digestion on minigel.
2. Extract cut template in an equal volume of 50 : 48 : 2 phenol : chloroform : isoamyl
alcohol. Spin down, transfer the upper layer to a fresh tube and extract with chloroform
: isoamyl alcohol.
3. Precipitate upper layer with 1/9 volume of 3M NaOAc and 2 volume ethanol at80°C.
Spin down at 4°C for 15 minutes. Wash pellet with 70% EtOH and spin again.
Resuspend pellet in 20µl of RNAsefree TE, and store at 4°C until required.
Important note: even very small amounts of DepC can inactivate RNA polymerase. I
have noted reductions in yield if I use DepC treated water to make RNAsefree TE,
even though the water was autoclaved before making the TE. I resuspend my cut
plasmid in Steve's AGDW, and also use it for reaction water. These details alone
increased my RNA yield from around 15µg per reaction to 60µg per reaction. (PMW)
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4. Set up the following reaction in 50µl total volume:
5x Stratagene synthesis buffer 10µl
0.1M DTT (RNAase free) 5µl
10mM NTPs/digoxygeninUTP 2.5µl
RNAsin 0.25µl (10 units)
RNA polymerase (T3, T7 or Sp6) 4.5µl (90 units)
DNA template 2.5µg
RNAse freewater to 50µl
Incubate at 37°C for 2 hours.
5. Remove 2µl for minigel sample. Add 20 units of RNAsefree DNAse and continue
incubation for a further 10 minutes at 37°C. Remove a second 2µl sample and check that
DNA has been degraded on a 1% TBE minigel.
6. Add 52µl of 'Stop' buffer to the reaction.
7. Separate unincorporated ribonucleotides on a Sephadex G50 spin column by spinning
for 2 minutes on setting 5 on the Anderson Lab benchtop centrifuge. (Modify as
necessary).(Noted by Ding: no need to do it)
8. Transfer the purified probe to a clean tube. Add 1/9 volume of 3M NaOAc, pH4.8 and
2 volumes of ethanol. Precipitate at 80°C for 10 minutes. Spin down at 4°C for 15
minutes, wash the pellet in 95% EtOH/5% DEPCwater and spin again for 5 minutes.
9. Resuspend the pellet in 50µl of an RNAsefree solution of 40mM NaHCO3 / 60mM
Na2CO3. Remove a 1µl sample for OD260 measurement. Incubate at 60°C for 35 minutes
to hydrolyse the probe into small fragments (between 200300 bp). 35 minutes works
fine for a 1kb probe. For other probe sizes, use the following formula:
The amount of time, t , for hydrolysis in 40mM NaHCO3 / 60mM Na2CO3 at 60°C is
given by:
(Starting length, kb) (Desired length, kb)
t = —————————————————————
(0.11) (Starting length, kb) (Desired length, kb)
10. Precipitate hydrolysed probe again as in (8) above. Resuspend the probe in
hybridisation buffer to a final concentration of 10µg/ml. N.B. Resuspend in a small
volume first, then make a final dilution.
11. Store probe at 20°C until required. Probes should stay stable for months on end.
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C: Sephadex G50 Spin Column
1. Suspend Sephadex G50 in distilled water. DEPCtreat overnight and autoclave. Let
Sephadex settle out, and replace water with RNAsefree 0.3M NaOAc pH 6.0/ 0.1% SDS.
2. Pack the base of a 3ml syringe with glass wool that has been siliconised with
"Sigmacote" and autoclaved. Handle wool with forceps heated in a gas jet.
3. Load 5ml of G50 slurry onto the column and spin down. (3 minutes at setting 5 on
Anderson Lab benchtop centrifuge modify as necessary).
4. Remove buffer from tube and replace with a clean Eppendorf tube. Column is now
ready for use.
D: Preabsorbing AntiDigoxygenin Antibody
1. Fix a series of rat/chick E12.5 E13.5 embryos in 4% paraformaldehyde for 2 hours at
room temperature. Use about 8 rat embryos for every ml of 1:200 antibody. Wash with
PBS and store at 20°C in methanol.
2. Rehydrate in:
75% MeOH : 25% H2O 5 minutes at room temperature
50% MeOH : 50% H2O " " "
25% MeOH : 75% H2O " " "
100% PTw " " "
3. Dissociate embryos with a syringe and incubate in 10% heat inactivated serum in PTw
for one hour at room temperature. Spin down and discard supernatant.
4. To the minced embryos add an equal volume of 1:200 antidigoxygenin antibody in
PTw containing 1% serum. Incubate for three hours at room temperature on a rotary
wheel.
5. Spin down the embryos, recover supernatant and dilute to 1:2000 final concentration
with PBT containing 20% serum.
N.B. Use a serum species compatible with the antibodies you are using. The Boehringer
antiDIG antibodies are made in sheep. Heat inactivate serum by incubating it at 55°C
for 30 minutes.
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E: Preabsorbing AntiDigoxygenin Antibody Embryo Powder Method
This is an alternative to the protocol above, which may be more convenient for small
batches of antibody.
1. Homogenise embryos of an appropriate age and species in a minimum volume of ice
cold PBS, by squirting through a 30mL syringe.
2. Add 4 volumes of ice cold acetone, mix well and incubate on ice for 30 minutes.
3. Spin pellet out at 10,000g for 10 minutes, wash with ice cold acetone and spin down
again.
4. Spread pellet out on filter paper, let it dry thoroughly and grind into a fine powder.
Store at 4°C. The yield is surprisingly low. Two dozen D8 chicks can yield 25mLs of
homogenized embryos, and 1.2g of powder.
To produce 2ml of preabsorbed antibody:
5. Weigh out 3mg embryo powder and mix with 0.5ml PBT.
6. Incubate at 70°C for 30 minutes. Vortex hard for 10 minutes.
7. Cool mixture on ice, add 5µl serum and 1µl antidigoxygenin antibody. Mix for 1 hour
at 4°C.
8. Spin down hard and dilute supernatant to 2ml with PBT containing 20% serum.
F: Fixation of Embryos for Sectioning
Embryos smaller than about E18 rat can be fixed by immersion in 4% paraformaldehyde
in 0.1M phosphate buffer. Larger animals (like newborn pups) must be fixed by
perfusion. Perfusion is necessary because fixative cannot simply diffuse into the tissues
of these larger, more developed animals in time to preserve morphology, mRNA, and
antigens. Perfusion requires opening the thoracic cavity of a living specimen,
puncturing the lower ventricle of the heart with a fine gauge butterfly needle, and
pumping fixative through the circulatory system. If you have never done this, have it
demonstrated for you the first time.
To make 20mL of 4% paraformaldehyde:
1. Weigh out 0.8g solid paraformaldehyde. Add to 10mL distilled water in a screwcap
type disposable tube. Add 20µl 10N NaOH. Place in the 68°C water bath for a few
minutes until the paraformaldehyde goes into solution.
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2. Add 15µl concentrated HCl (12.7N). Cool on ice.
3. Add 10mL 0.2 M phosphate buffer pH 7.4. 0.2M phosphate buffer must be kept
sterile: contamination will result in your signal being completely destroyed.
4. Fix animals at 4°C for 4 to 24 hours. Change solutions to 15% sucrose in 0.1M PB.
Incubate at 4°C until the animals sink.
5. Wash animals in OCT. Place in mounting form in fresh OCT. Freeze on dry ice. Store
blocks at 80°C for up to a year.
Some people feel very young animals, such as D3D4 chicks, must be mounted in
gelatin for good histology. I have found the sharpness of the blade becomes the most
important factor if the tissue is fixed properly.
Paraformaldehyde that has been made up in a large batch and stored for more than a
day does not necessarily fix tissue properly. If you are storing blocks at 80°C and find
you lose signal, or morphology, over time, you are not fixing your tissue properly. Try
using fresh fix. (PMW)
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Solutions
Stop Buffer
1% SDS
20mM EDTA
20mM Tris pH7.5
100mM NaCl
PK Buffer for sections:
50mM TrisHCl pH 7.5
5mM EDTA
1M Triethanolamine, pH 8.0:
Add 66.5 Triethanolamine and 20ml conc. HCl to 413.5ml DEPCwater in an
RNAsefree bottle.
PTw:
1xPBS
0.1% TWEEN20
PBT:
1xPBS
2mg/ml BSA
0.1% Triton X100
100x Denhardt's Solution
2% BSA (ICN 810661)
2% Polyvinylpyrrolidone (PVP40)
2% Ficoll 400
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Make a slurry in DEPCwater and dilute.
Hybridisation Solution:
For 50ml
50% Formamide 25ml
5x SSC 12.5ml 20xSSC
0.3mg/ml Yeast tRNA 0.3ml of 50mg/ml in DepCH2O
100µg/ml Heparin 50µL of 100 mg/mL in DepCH20
1xDenhardt's Solution 0.5ml 100x
0.1% Tween 20 0.5mL 10% Tween in DepCH20
0.1% CHAPS 0.5mL 10% CHAPS in DepCH20
5mM EDTA 0.5mL of 0.5M EDTA pH 8.0
All components should be RNAse free
Cheaper Hybridisation Solution for Whole Mounts:
For 100ml
50% Formamide 50ml
5xSSC 25ml 20xSSC
20µg/ml Yeast tRNA 0.1 of 20mg/ml in DepCH2O
100µg/ml Heparin 10mg
0.1% Tween 20 1mL 10% Tween in DepCH20
0.1% CHAPS (Sigma C3023) 1mL 10% CHAPS in DepCH20
5mM EDTA 1mL 0.5M EDTA pH 8.0
All components should be RNAse free
NBT:
75 mg/ml Nitro blue tetrazolium in 70% dimethyl formamide and 30% water.
Hai suggests making a stock of 30mg/mL in 70% formamide and adjusting the
amount added accordingly.
20
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BCIP:
50 mg/ml 5bromo4chloro3indoyl phosphate in 100% dimethyl formamide
TBS:
500mM NaCl
20mM Tris, pH 7.5
TBST:
500mM NaCl
20mM Tris, pH 7.5
1% Tween 20
MEMFA:
0.1M MOPS pH 7.5
2mM EGTA
1mM MgSO4
3.7% Formaldehyde
Make a 10x stock of the salts and add fresh formaldehyde each time.
Alkaline Phosphatase Buffer:
100mM Tris, pH 9.5
50mM MgCl2
100mM NaCl
0.1% TWEEN 20
5mM Levamisole add fresh each time.
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N.B. This buffer should always be made up fresh each time from its components it
tends to acidify quite quickly. (Tris pH 9.5 mixed with the MgCl2 is the problem. Mix
the other three components with water, and add the Tris right before use. HW)
Reagents, Glassware and Apparatus
The following solutions can be made up in untreated bottles. Treat with 0.05% DEPC
overnight at 37°C and then autoclave:
EDTA, NaCl, MgCl2, NaHCO3 / Na2CO3, NaOAC
The following solutions cannot be autoclaved. Make these up in DEPCtreated water in
glass bottles:
Tris, SDS, any buffers with Tween, Triton or CHAPS
The following solutions cannot be autoclaved. Make up in sterile 50mL tubes:
Hybridisation buffer, Denhardt's
The following solutions do not need to be RNAse free:
TBS, TTBS, PBT, BCIP, NBT, MEMFA, TrisImidazole buffer
PTw for whole mounts and cultured cell in situs needs to be made with DEPCPBS. For
posthybridisation washes, this is not necessary.
RNAse free forceps and spatulas should be flamed in a gas jet just prior to use.
Glass boats, jars, and slide mailers cannot be autoclaved. Before each experiment, soak
them overnight in water containing 0.05% DEPC at 37°C. Wash them twice in DEPC
treated water before use.
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Ordering Information
Reagent Company Catalog Amount Used in
number
antidigoxygenin Fab Boehringer 1 093 274 antibody
Mannheim
antifluorescein Fab Boehringer 1 426 338 twoprobe double
Mannheim label antibody
3aminopropyl Sigma A3648 100 mL subbing slides
triethoxysilane (TESTA)
AP color development BioRad 1706539 300mg AP reaction mix
reagent BCIP
AP color development BioRad 1706532 600mg AP reaction mix
reagent NBT
Bovine serum albumin ICN 810661 hybridization buffer
Bovine serum albumin Sigma A3912 250 g PBT
CHAPS Sigma C3023 hybridization buffer
Ficoll 400 hybridization buffer
Formamide Fluka 4767 250mL hybridization buffer
Heparin Sigma H3393 10,000 hybridization buffer
units
INT/BCIP stock solution Boehringer 1 681 460 3 mL twoprobe double
Mannheim label
Levamisole Sigma L9756 10g AP reaction mix
Polyoxyethylene sorbitan Sigma P1379 hybridization buffer,
monolaurate (Tween20) AP reaction mix
Polyvinylpyrrolidine (PVP hybridization buffer
40)
Proteinase K Sigma P6556 25mg prehybe
Ribonuclease A Sigma R5503 100mg SSC washes
Sephadex G50 Pharmacia 17004502 100g Spin column
Sheep serum (lamb) Gibco 16070013 100mL antibody buffer
SigmaCoat Sigma SL2 100mL Spin column
SuperFrost/Plus microscope Fisher 1255015 slides that don't need
slides subbing
Type XSA transfer Sigma R8759 10,000 hybridization buffer
ribonucleic acid units
Probe synthesis
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