Radiactive In Situ Hybridisation

Contents 1 Protocol for Radioactive 33P In Situ Hybridisation 1.1 DAY ONE: 1.2 Part A: Making the probe 1.3 Part B: Making the Hybridisation Buffer 1.4 Part C: Hybridise the slides 1.5 DAY TWO: 1.6 Partn A: Post- hybridisation washes 1.7 Part B: Developing film 2 Soluions needed 2.1 To be made prior to Day 1: 2.2 To be made on the day of use:

Protocol for Radioactive 33P In Situ Hybridisation

Clare Walton, Nottebohm lab, Rockefeller University

Prior to Day 1- DEPC-treat solutions and autoclave them. See solutions at end of protocol. Purify and linearise template plasmid DNA if needed. Order radioactive 33P-UTP.

DAY ONE:

Part A: Making the probe

Reagents needed:

• Template DNA, linearised
• Transcription optimised buffer, 5x
• RNA Polymerase
• RNasin
• ATP, GTP and CTP
• 33P-labelled UTP
• DTT, 100mM
• Nuclease-free H2O

1- Wash all glassware with SDS on day 1 and wipe down bench with RNase away.

2- Thaw reagents for probe synthesis (transcription buffer, ribonucleotides, DTT etc)

3- Dry down 5-7μl hot UTP in concentrator. Pipette solution into 1.5ml epidorfs and spin with lids open for 10 minutes.

4- Make up transcription mix as follows (for 50μl probe):

2 ul 5x buffer

1 ul DTT (100mM)

0.25 ul RNasin

0.5 ul ATP (10mM)

0.5 ul CTP (10mM)

0.5 ul GTP (10mM)

0.5 ul Linearised DNA template

0.5 ul Sp6 RNA Pol

4.25 ul dH2O

5- Add 10μl transcription mix to the dried-down hot UTP and pipette to resuspend. Put the tubes in the radioactivity pig and place in the 37°C incubator for 2 hours. Proceed to step B: prepare the hybridisation buffer.

6- Stop reaction with 90μl TE .

7- Add 2μl tRNA (10mg/ml) and 100μl of 5M Ammonium acetate. Flick tube to mix.

8- Add 600μl of 100% EtOH from the -20°C freezer and vortex tube for 10 secs.

9- Place tube in pig in the -80°C freezer to precipitate for 30-60 mins.

10- Spin tube at 4°C at max speed in a benchtop centrifuge for 20 mins.

11- Pipette off the S/N. Pellet should be hot.

12- Wash pellet in 70% EtOH. Add EtOH and leave for 10 secs, then pipette off. Do not resuspend or mix. Leave cap open to allow pellet to air dry (about 10 mins).

13- Resuspend dry pellet in 50-55μl TE. Add TE and leave for 15 mins before resuspending by trituration. Once probe is made, it can be stored at -20°C for about 5 days. Use at 1-2μl per slide, depending on the scintillation reading.

Scintillation counter- measure radioactivity of probe in the scintillation counter. Add 1μl to one measure of scintillation fluid in a glass scintillation vial. Reading should be between 1,000,000 and 2,000,000 cpm/μl.

Part B: Making the Hybridisation Buffer

1- Turn on the incubator to 65°C to dissolve the dextran sulphate.

2- Get Denhardts, tRNA and poly A reagents out of freezer to thaw.

3- Turn on the oil hybridisation bath about 2 hours prior to hybridisation to allow the oil to heat to 65°C.

4- Make dextran sulphate solution. Add 2 g dextran sulphate to in 10 ml formamide in a 15ml falcon tube. Add quickly and immediately invert to stop a plug forming in the tip. Heat at 65°C for about 30 mins until dissolved.

5- Make 2x Hyb buffer in a 50ml falcon tube as follows:

4 ml 10x SET

400 ul 50x Denhardts

200 ul 20% SDS

500 ul tRNA (10mg/ml)

250 ul polyA (2mg/ml)

4.65 ml DEPC H2O

Add reagents but do not vortex as it will introduce air bubbles. Mixture may be cloudy. Warm to 40-50°C briefly (in a water beaker over a Bunsen burner) to fully mix solution. It should become clear.

6- Add 10ml formamide/dextran solution to 10ml 2x hyb buffer and gently vortex. Heat the mixture to 40-50°C briefly to mix.

7- Aliquot the amount you will need and put it in the heat block at 60°C. The rest can be stored in the fridge if to be used within a few days. If not, store at -20°C.

Part C: Hybridise the slides

This protocol uses no pre-hybridisation incubation and is for fresh frozen unfixed tissue.

1. Turn oven to 37°C.
2. Remove slides from the -80°C freezer and put into grey plastic slide racks.
3. In the green container, add 200ml 4% PFA in PBS from the fridge. Leave for 15 mins at RT on a shaking platform.
4. Wash with PBS, three times at 5 mins each.
5. Immerse in 200ml TEA for 2 mins on the shaking platform.
6. Pour the TEA back into the cylinder and add 500μl per 200ml of acetic anhydride.
7. Cover cylinder with parafilm, vigorously invert to mix and pour back over slides for 10 mins with shaking.
8. Wash with PBS for 5 mins.
9. Dehydrate slides through ethanols. 2 mins each in 50, 70 and 95% solutions.
10. Place rack of slides in the oven at 37°C with the door ajar for 15 mins to dry the slides.
11. At this point, add probe to hybridisation buffer (1-2μl per slide ). Vortex and place back into heating block to get rid of bubbles.
12. Add probe in hyb buffer to slides. Pipette 65-90μl along the edge of the slide trying not to introduce bubbles at the end. Place a coverslip along the edge until it catches the liquid and then drop down onto the slide.
13. Stack finished slides horizontally in a metal slide rack and place the rack in the 65°C oil bath overnight. Place the rack in the oil slowly and at an angle to allow air bubbles to escape.

DAY TWO:

Partn A: Post- hybridisation washes

1. Turn on the water bath to 37°C and put in the RNase buffer to warm in the plastic slide containers used with RNase. 200ml buffer per rack of slides.
2. Make 2 litres 2x SSC and warm 200ml per rack to 37°C in a bottle in the water bath.
3. Make 1.2 litres 0.1x SSC and warm to 60°C on a heating block with a stir bar in a 2 litre beaker.
4. Get the RNase out of -20°C freezer to thaw. Be careful to change gloves after touching, do not put on bench and only use with filter tips.
5. Fill glass dishes with 200ml 2xSSC (one per rack) and heat to about 55°C in the microwave (3-4mins).
6. Fill 2 glass dishes with chloroform in the fume hood.
7. Remove the slide rack from the oil bath slowly and put into the first chloroform wash. Use the wire handle to rapidly dunk the slide rack up and down about 20 times. Repeat in the second chloroform wash. (Coverslips should stay on).
8. Move the rack to the warm 2xSSC and dunk a few times. Leave in the 2xSSC for the coverslips to come off while disposing of chloroform.
9. Pour hot chloroform waste into the radioactive waste bottle using a funnel. Spray the glass dishes with Count-off and wipe clean. Test for remaining 33-P with a Geiger counter.
10. Remove coverslips in the radioactivity hood. Use tweezers to pick up each slide and gently slide off the coverslip. Place the slide in 2xSSC at RT in plastic containers and dispose of the coverslip in an old 50ml falcon tube. Pour the warm SSC into the radioactive waste but from now on the SSC washes can be poured down the sink.
11. Move racks into a 2 litre beaker full of RT 2xSSC with a stir bar for 15 mins.
12. Add RNase A to warm RNase buffer: 0.5ml of 10mg/ml stock to each 200ml container of buffer (one per rack).
13. Put slide rack in RNase at 37°C for 30 mins.
14. Pour out RNase and do a quick wash with warm 2x SSC from water bath.
15. Move rack into 0.1x SSC at 60°C with a stir bar for 1 hour.
16. Return rack to plastic container and incubate with 35% EtOH with Ammonium acetate for 2mins.
17. Repeat with 70% EtOH and 95% EtOH, both containing Ammonium acetate.
18. Place rack in the 37°C incubator to dry with the door ajar for at least 15 mins.
19. Once dry, lie slides in a film cassette with tissue facing up. In the dark, place the film with the matt side down over the slides and close the cassette.
20. The exposure time will vary. Use the Geiger counter to estimate how long it will take.

Part B: Developing film

1. With lights off, place the exposed film in the metal frame.
2. Immerse in the developer for 3 mins. Do not remove the cover of the developer compartment when the light is on.
3. Move the film to water for 30 secs.
4. Put into fix for 5 mins.
5. Wash in water for 5 mins. The lights can be turned on at this point.
6. Hang the film up to dry above the sink.

Soluions needed

To be made prior to Day 1:

DEPC treatment (to remove RNases): Use at 0.1% (0.5ml per 500ml solution) in the fume hood. Be Careful, DEPC is a carcinogen. Shake thoroughly to mix and leave overnight in hood with lid loose for smell to reduce. Autoclave the following day for a minimum of 45 mins to remove DEPC.

10x DEPC-treated PBS – 1 litre

2 g KCl

2 g KH2PO4

11.52 g Na2HPO4

80 g NaCl

Make with dH2O, pH to 7.4, divide into 2 bottles to avoid spillover in autoclave, DEPC treat O/N and autoclave for 45 mins.

0.1 M TEA (Triethanolamine) pH 8.0, for 500ml

6.7ml TEA into 400ml dH2O. pH to 8.0 with HCl and make up to 500ml with dH2O. DEPC treat overnight and autoclave.

5M NaCl

146.14g NaCl per 500ml dH2O. DEPC treat overnight and autoclave.

0.5M EDTA pH8.0

14.6g EDTA disodium salt in 100ml dH2O. Add 70ml and pH with conc NaOH. Will only begin to dissolve as pH approaches 8.0. Make up to 100ml once dissolved. DEPC treat overnight and autoclave.

20% SDS

Do not autoclave. Use H2O previously DEPC-treated and autoclaved. 10g SDS in 50ml DEPC-H2O. Make in fume hood. pH to 7.2.

1M Tris pH 7.8

Do not DEPC-treat Tris solutions. Use DEPC-treated and autoclaved H2O. 12.1g Trizma base per 100ml DEPC-H2O. Add 70ml and pH to 7.8 with conc HCl. Make up to 100ml.

1M Tris pH 7.5

For RNase buffer so does not need to be RNase free. 12.1g Trizma base per 100ml dH2O. Add 70ml and pH to 7.5 with conc HCl. Make up to 100ml.

20x SET (salt, EDTA and tris)

6ml of DEPC-treated 5M NaCl (final conc 3M)

400μl of DEPC-treated 0.5M EDTA pH8.0 (final conc 20mM)

4ml of RNase-free 1M Tris pH7.8 (final conc 400mM)

20x SSC pH 7.0

175.3g NaCl and 88.2g Na citrate per 1 litre dH2O. Autoclave.

RNase buffer, 500ml

50 ml 5M NaCl

5 ml 1M Tris pH7.5

1 ml 0.5M EDTA

444 ml dH2O

EtOH with 300mM NH4 acetate, 35%, 70% and 95%.

For 500mls:

• • 35%: 11.65g NH4Ac, 325ml dH2O, 175ml 100% EtOH
  • 70%: 11.65g NH4Ac, 150ml dH2O, 350ml 100% EtOH
  • 95%: 11.65g NH4Ac, 25ml dH2O, 475ml 100% EtOH

To be made on the day of use:

Dextran sulphate/Formamide 2X solution

Add 2g dextran sulphate to 10ml 100% formamide in a 15ml falcon tube and rapidly invert to stop an insolulable clump forming. Heat to 65°C in the rotating incubator for 30mins until fully dissolved. To store: -20°C

2X Hybridisation buffer (for 10ml)

4 ml 20x SET

400 ul 50x Denhardts

200 ul 20% SDS

500 ul tRNA (10mg/ml)

250 ul polyA (2mg/ml)

4.65 ml DEPC dH2O

Add to a 15ml conical flask and heat to 50°C until clear. Mix 1:1 with dextran sulphate/formamide before use but store separately at -20°C. Note: tRNA is Sigma R7876 500 units Type V Wheat germ 9014-25-9. PolyA is Sigma P9403 25mg

RNase solution

RNase stock is 10mg/ml. Add 1ml to 500ml RNase buffer.

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