Anderson Lab In Situ Hybridization Protocols

March 1997

These protocols describe non-radioactive methods for in situ

hybridization on frozen sections, whole mount embryos and on cultured

cells. They have been freely adapted and modified from the protocols of

Richard Harland, David Wilkinson, Domingos Henrique, Andy McMahon,

Tony Campagnioni, and others. This protocol is modified from the

November 1995 version previously circulated.

1

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 DEPC-PBS and three changes of DEPC-water 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: Pre-Treatment of Sections

N.B. All reagents and mailers in the following steps must be RNAse-free.

In this section, steps 6 and 7 (the acetylation) is vital for some probes,

but raises the background for others. Some probes, like SCG-10, Brn-3

and neurofilament, work best without acetylation. Other probes, like

GATA-2, 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 DEPC-PBS at room temperature for 20

minutes.

3. Wash twice in DEPC-PBS at room temperature for 5 minutes.

4. Treat slides with 50µg/ml Proteinase K in PK buffer at room temperature

for between 8-15 minutes depending on the age of the embryo.

5. Wash once in DEPC-PBS at room temperature for five minutes. Fix in 4%

paraformaldehyde in DEPC-PBS for 15 minutes.

6. Rinse once in DEPC-water.

7. Place slides in an RNAse-free glass trough with a stir bar. Add 250ml

0.1M RNAse-free triethanolamine-HCl 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 DEPC-PBS at room temperature for five minutes.

2

9. Prehybridise for 3-4 hours at 60°C. Replace with 1-2µg/ml of probe and

continue incubation for a further 12-16 hours. These steps should be

performed in the 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 RNAse-free 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% heat-inactivated 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 pre-absorbed anti-digoxygenin 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.

3

4. For every ml of Alkaline Phosphatase buffer, add 1µl of NBT and 3.5µl of

BCIP, and develop in the dark for between 2-20 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.

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 1-2µg/ml each.

C: Staining.

The following steps are performed in slide mailers.

4

1. Sections are incubated with either anti-fluorescein-AP Fab fragments or

anti-digoxygenin-AP Fab, pre-absorbed and at the appropriate dilution in

20% sheep serum in PBT, at 4C overnight. Stain the weaker probe first.

2. After washing, stain with NBT/BCIP as in the normal protocol, until

staining reachs the desired intensity.

3. Wash three times in PBS at room temperature for 5 minutes each to

remove substrates.

4. Incubate in TE (100mM Tris-HCl pH 7.5, 50mM EDTA) at 85C for 10

minutes to inactivate alk-phos. Use the 68C water bath turned up to 85C.

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% heat-inactivated sheep serum in PBT at room

temperature for 30 minutes.

8. Incubate slides with the other antibody at 4°C overnight. If you used

anti-fluorescein originally, use anti-digoxygenin 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 2-20 hours, depending on the

abundance of the RNA.

N.B. Anti-digoxygenin-AP Fab final concentration is 1:2000. Anti-

fluorescein-AP 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)

5

A: Pre-Treatment 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.

2. Fix in 4% paraformaldehyde in DEPC-PBS at room temperature for 20

minutes.

3. Wash twice in DEPC-PBS at room temperature for 5 minutes.

4. Prehybridise for 3-4 hours at 60°C. Replace with 1-2µg/ml of probe and

continue incubation for a further 12-16 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 tissue-culture grade

D-PBS 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 D-PBS.

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 pre-treated with

1µg/mL proteinase K and hybridized at 60C 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 mouse-HRP, in this case) in

1% goat serum, 1% BSA, 0.1% Triton in TC D-PBS.

6

7. Wash three times in PBT at room temperature for 5 minutes each.

8. Wash once in PBT at 4C 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 nickel-DAB 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% heat-inactivated sheep serum in PBT at room

temperature for one hour.

Now follow the normal protocol for visuallization of digoxygenin (or

fluorescein.)

Very important: Fixing the slides in MEMFA eliminates the Ni-DAB 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 68C or 72C.

If you are re-using the 20% sheep serum block or anti-digoxygenin 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

high-stringency step strips the probe off the sections. It is necessary to

reduce background, but I have found that with clean probes, the high-

stringency washes can be pared back to 25 minutes and this improves

signal. The same is true for the RNAse A step. (PMW)

7

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.

8

II: Whole Mount In Situ Hybridization.

Embryos should be dissected free of any extra-embryonic membranes,

fixed in 4% paraformaldehyde in DEPC-PBS for 2 hours at room

temperature (or 4°C overnight), washed in DEPC-PBS, 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 5-6 ml liquid. The tubes are rocked gently on a rotating

platform to allow thorough exchange of solutions.

A: Pre-Treatment 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 5-60 minutes. This

is a critical step, as over-digestion will destroy the embryos, and under-

digestion 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 re-fixed.

5. Rinse twice gently in PTw. Re-fix 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

9

too little than too much. Replace with 1 ml hybridisation mix. Remove this

mixture after the embryos sink, and replace with fresh hybridisation mix.

8. Pre-hybridise 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 pre-warmed (70°C) hybridisation mix for five

minutes each in the same tube.

2. Wash twice with pre-warmed hybridisation mix for 30 minutes each at

70°C.

3. Wash once with a 1:1 mixture of hybridisation mix and TBST (pre-

warmed) at 70°C for 20 minutes.

4. Wash twice with TBST at room temperature for five minutes each.

5 Incubate embryos in 50 g/mL RNaseA in TBST at 37C 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 60C8. 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 pre-absorbed anti-digoxygenin 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.

10

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 2-20 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 4C 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.

11

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: Pre-Treatment of Cells

1. Fix in 4% paraformaldehyde in DEPC-PBS at room temperature for 10

minutes.

2. Wash three times in DEPC-PTw 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 DEPC-PTw for 5 minutes each.

5. Digest with 10 ug/ml of Proteinase K in DEPC-PTw for 10-30 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 DEPC-PTw.

7. Fix again in 4% paraformaldehyde in DEPC-PBS for 15 minutes.

8. Wash three times in DEPC-PTw 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 DEPC-SSC for five minutes at room temperature.

11. Pre-hybridize for 6 hours at room temperature or 3 hours at

hybridization temperature. Remove pre-hyb 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 tight-sealing 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.

12

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 anti-digoxygenin 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 pre-absorbed 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 2-36 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.

13

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 (3-aminopropyltriethoxysilane;

Sigma A-3648) in dry acetone for 5 minutes.

4. Wash in 2 changes of acetone and three changes of DEPC-water. 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 maxi-prep quality plasmid DNA with a five-

fold excess of an appropriate restriction enzyme for 2 hours. Check

digestion on mini-gel.

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 at-80°C. Spin down at 4°C for 15 minutes. Wash pellet with 70%

EtOH and spin again. Resuspend pellet in 20µl of RNAse-free TE, and store

at 4°C until required.

Important note: even very small amounts of Dep-C can inactivate RNA

polymerase. I have noted reductions in yield if I use Dep-C treated water to

make RNAse-free 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)

14

4. Set up the following reaction in 50µl total volume:

5x Stratagene synthesis buffer 10µl

0.1M DTT (RNAase free) 5µl

10mM NTPs/digoxygenin-UTP 2.5µl

RNAsin 0.25µl (10 units)

RNA polymerase (T3, T7 or Sp6) 4.5µl (90 units)

DNA template 2.5µg

RNAse free-water to 50µl

Incubate at 37°C for 2 hours.

5. Remove 2µl for mini-gel sample. Add 20 units of RNAse-free 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 mini-gel.

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%

DEPC-water and spin again for 5 minutes. 9. Resuspend the pellet in 50µl of an RNAse-free 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

200-300 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.

15

11. Store probe at -20°C until required. Probes should stay stable for

months on end.

16

C: Sephadex G-50 Spin Column

1. Suspend Sephadex G-50 in distilled water. DEPC-treat overnight and

autoclave. Let Sephadex settle out, and replace water with RNAse-free

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 G-50 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: Pre-absorbing Anti-Digoxygenin 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 anti-digoxygenin

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.

17

N.B. Use a serum species compatible with the antibodies you are using.

The Boehringer anti-DIG antibodies are made in sheep. Heat inactivate

serum by incubating it at 55C for 30 minutes.

E: Pre-absorbing Anti-Digoxygenin 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 pre-absorbed 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 anti-digoxygenin 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

18

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

screw-cap type disposable tube. Add 20µl 10N NaOH. Place in the 68°C

water bath for a few minutes until the paraformaldehyde goes into solution.

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 4C for 4 to 24 hours. Change solutions to 15% sucrose in

0.1M PB. Incubate at 4C until the animals sink.

5. Wash animals in OCT. Place in mounting form in fresh OCT. Freeze on

dry ice. Store blocks at -80C for up to a year.

Some people feel very young animals, such as D3-D4 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 -80C and find you lose signal, or morphology, over time, you are

not fixing your tissue properly. Try using fresh fix. (PMW)

19

SolutionsStop Buffer

1% SDS

20mM EDTA

20mM Tris pH7.5

100mM NaCl

PK Buffer for sections:

50mM Tris-HCl pH 7.5

5mM EDTA

1M Triethanolamine, pH 8.0:

Add 66.5 Triethanolamine and 20ml conc. HCl to 413.5ml DEPC-water

in an RNAse-free bottle.

PTw:

1xPBS

0.1% TWEEN-20

PBT:

1xPBS

2mg/ml BSA

0.1% Triton X-100

100x Denhardt's Solution

2% BSA (ICN 810661)

2% Polyvinylpyrrolidone (PVP-40)

2% Ficoll 400

Make a slurry in DEPC-water and dilute.

20

Hybridisation Solution:

For 50ml

50% Formamide 25ml

5x SSC 12.5ml 20xSSC

0.3mg/ml Yeast tRNA 0.3ml of 50mg/ml in DepC-H2O

100µg/ml Heparin 50L of 100 mg/mL in

DepC-H20

1xDenhardt's Solution 0.5ml 100x

0.1% Tween 20 0.5mL 10% Tween in DepC-

H20

0.1% CHAPS 0.5mL 10% CHAPS in

DepC-H20

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 DepC-H2O

100µg/ml Heparin 10mg

0.1% Tween 20 1mL 10% Tween in DepC-

H20

0.1% CHAPS (Sigma C-3023) 1mL 10% CHAPS in

DepC-H20

5mM EDTA 1mL 0.5M EDTA pH 8.0

All components should be RNAse free

NBT:

21

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.

22

BCIP:

50 mg/ml 5-bromo-4-chloro-3-indoyl 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 EGTA1mM MgSO4

3.7% Formaldehyde

- Make a 10x stock of the salts and add fresh formaldehyde each time.

Alkaline Phosphatase Buffer:

100mM Tris, pH 9.550mM MgCl2

100mM NaCl

0.1% TWEEN 20

5mM Levamisole - add fresh each time.

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

23

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 DEPC-

treated 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, Tris-Imidazole buffer

- PTw for whole mounts and cultured cell in situs needs to be made with

DEPC-PBS. For post-hybridisation 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.

24

Ordering InformationReagent Company Catalog

numberAmount Used in

anti-digoxygenin Fab Boehringer Mannheim

1 093 274 antibody

anti-fluorescein Fab Boehringer Mannheim

1 426 338 two-probe double label antibody

3-aminopropyl triethoxysilane (TESTA)

Sigma A-3648 100 mL subbing slides

AP color development reagent BCIP

BioRad 170-6539 300mg AP reaction mix

AP color development reagent NBT

BioRad 170-6532 600mg AP reaction mix

Bovine serum albumin ICN 810661 hybridization bufferBovine serum albumin Sigma A-3912 250 g PBT CHAPS Sigma C-3023 hybridization bufferFicoll 400 hybridization bufferFormamide Fluka 4767 250mL hybridization bufferHeparin Sigma H-3393 10,000

unitshybridization buffer

INT/BCIP stock solution Boehringer Mannheim

1 681 460 3 mL two-probe double label

Levamisole Sigma L-9756 10g AP reaction mixPolyoxyethylene sorbitan

monolaurate (Tween-20)Sigma P-1379 hybridization buffer,

AP reaction mixPolyvinylpyrrolidine

(PVP-40)hybridization buffer

Proteinase K Sigma P-6556 25mg pre-hybeRibonuclease A Sigma R-5503 100mg SSC washesSephadex G50 Pharmacia 17-0045-02 100g Spin columnSheep serum (lamb) Gibco 16070-013 100mL antibody bufferSigmaCoat Sigma SL-2 100mL Spin columnSuperFrost/Plus microscope

slidesFisher 12-550-15 slides that don't need

subbingType X-SA transfer

ribonucleic acidSigma R-8759 10,000

unitshybridization buffer

Probe synthesisReagent Company Catalog number Amount0.1M DTT Promega P117B 250L5x transcription buffer Promega P118B 500LDIG-RNA labeling mix Boehringer Mannheim 1 277 073 40LFluorescein-RNA labeling mix Boehringer Mannheim 1 685 619 40LRNAse inhibitor Boehringer Mannheim 799 017 2000 unitsT3 RNA polymerase Promega P208C 1000 unitsT7 RNA polymerase Promega P207C 1000 units

25

Sp6 RNA polymerase Promega P108B 1000 unitsDNAse I, RNAse free Boehringer Mannheim 776 785 10,000 units

26