Whole mount in-situ Based on the Ronna’s protocol

Whole mount In Situ Hybridization

I. In vitro transcription using DIG labeling (Mia)

Plasmid linearization

Linearization: 20mg DNA restricted with the appropriate enzyme (50U) in 100ul

Incubate 2h / O.N and run on agarose gel to see full digestion


Remember to plan restriction of antisense for the probe and sense for the control

Use 5’ protruding termini or blunt.

Extract DNA

·  Add 100ml of RNase free water

·  Add 1 volume (200ml) of chloroform/phenol 1:1 to the restricted DNA [1]

and mix well

Spin 5 min X 2,000rpm

·  Remove supernatant to a new tube

·  Optional – add 200ml chloroform, mix well and spin again

·  Remove supernatant to a new tube

·  Add 20ml (10% from total volume) 3MNaAC (pH=5, DEPC treated)

·  Add 400ml (2 volumes) 100%ice cold EtOH - mix

·  15’X dry ice in EtOH bath

·  Spin 10’X4°c X max speed

·  Remove EtOH and add 800ml 70% ice cold EtOH

·  10’X dry ice in EtOH bath

·  Spin 10’X4°c X max speed

·  Resuspend to 1mg/ml in ddw (DEPC-treated or better with Nuclease free water)

·  Run 0.5ml on 1% agarose gel

Dig cRNA labeling

Mix the reagents in the following order at R.T:

RNase free water / 23ml
10X transcription buffer / 4ml
0.1M DTT / 4ml
Nucleotide mix / 4ml
RNase inhibitors / 1ml
RNA polymerase (10U/ml) / 2ml
Add 2ml (2mg) of linearized DNA template

Incubate 2hX 37°c

Take 0.5ml for gel check. The RNA band should be ten fold brighter than the DNA band

DNase treatment

2ml DNase I RNase free

1ml RNase inhibitors (20U)

Incubate 15-30’ X 37°c

Inactivation 10’ X 65°c

RNA purification

·  Add RNase free water - 180 ml or RNase free water 100ml

·  add 10% volume 3M NaAC (in DEPC) - 20 ml 4M LiCl 4ml - mix

·  add 3 volume of ice cold 100% EtOH - 600 ml 100%EtOH 300ml

mix well & place in -20°c X 30’ (or dry ice)

* Can precipitate O.N.

·  Centrifuge 12,000 rpm X 4°c X 10’

·  Wash with 600ml of 70% ice cold EtOH

·  Remove supernatant – air dry & resuspend pellet with 40ml RNase free water

·  Run 0.5ml on 1% agarose gel and photocopy immediately

·  Store plasmid in -200c / aliquots in -70°c.

- Alternatively; cleaning on G-50 column or by gel extraction if more than 1 band is seen

Check the phenol color – shouldn’t be brown (EZ-RNA kit has phenol-chloroforme solution)

II. Tissue Prep for ISH

A. Dissection

  1. Dissect embryos in PBS (remove membrane as much as possible)

Use tools treated with RNase away and disposable bulbs

From E9.5 punctate mylencephalon and make small hole in the heart and telencephalon

2. Fixation in 10 ml fresh 4% paraformaldehyde at 4°c X O.N X gentle rocking

B. Dehydration ~1h

24 hours after fixation

Use 50 ml tube with 20 ml solution of:

·  PBT X 5’X 4°c - X 2

·  25% methanol in PBT 5’X R.T

·  50% methanol in PBT 5’X R.T

·  75% methanol in PBT 5’X R.T

·  100% methanol 5’X R.T - X 2

Can store at -20°c as long as a month

C. Rehydration ~1h (in the experiment day)

Use 50 ml tube with 50 ml solution of (diluted with in PBT):

·  75% methanol in PBT 5’X R.T

·  50% methanol in PBT 5’X R.T

·  25% methanol in PBT 5’X R.T

·  PBT X 5’X 4°c - X 2

III. Pre-treatments/Deproteination ~1h40min (Ronna)

1. Treat with 10 µg/ml proteinase K in PTW for 10 min[2]

2. Wash 30 sec in 2 mg/ml glycine in PBT (fresh) X R.T

Wash 10’ in 2 mg/ml glycine in PBT (fresh) X R.T

3. Carefully rinse with PTW 2X 5 min

4. Incubate in Detergent Mix 2X 15 min, then rinse 2X 5 min PTW

5. Post-fix in 4% PFA+0.2% Glutraldehyde for 20 min

6. Rinse with PTW x 5 x shaker

IV. Hybridization – 1h30min until the o/n incubation

1. Rinse in 1:1 PTW to hybridization mix and let cochleae settle

2. Rinse in 1 ml hybrid mix and let cochleae settle

3. Incubate horizontally at 65ºC on shaker in 1ml fresh hyb mix for 1 hour

4. Add 1ml fresh pre-warmed hyb mix and 1µg (varies) DIG-labeled probe

5. Incubate horizontally at 65ºC on shaker overnight (O/N)

------End of day #1------

V. Post-Hybridization Washes

1. Rinse 2X in pre-warmed hyb mix

2. Wash 2X 30 min at 65ºC with 1.5ml pre-warmed hyb mix

3. Wash 10 min at 65ºC with 1:1 hyb mix to MABT pre-warmed

4. Rinse 2X with 1.5ml MABT x 5’ x 65ºC

5. Wash 15 min in 1.5ml MABT x 65ºC

**optional: incubate 1 hr with 1.5 ml MABT and 2% Boehringer Blocking Reagent (BBR), then add 2% BBR to step 6 and step 7.

6. Incubate 1 hour in MABT with 20% heat-treated sheep serum horizontally at 65ºC on shaker

7. Incubate 3 hours RT or O/N at 4ºC on shaker in MABT, 20% sheep serum, and 1/1000 dilution AP-anti-DIG antibody

------End of day #2------

VI. Post-Antibody Washes

1. Rinse 3X with MABT x 5’ x R.T x shaker

-Transfer tissue into glass vials

2. Wash 2X 45 min with MABT at Room Temperature (RT) while shaking horizontally

VII. Histochemistry

1. Wash 2X for 10 min in 5ml NTMT (fresh)

2. Incubate at RT in the dark while shaking with NTMT-BCIP-NBT mix

* purple color will develop (10 min- >3 days)

*change solution every few hours until color develops

3. Remove solution when color is developed

4. Wash for 10 min with 5ml NTMT

5. Wash for 10 min with PBS

*can be stored in PBS at 4ºC

VIII. Bleaching (Optional)

-Used to minimize background color

1. Warm cochleae to RT if originally at 4ºC

2. Replace PBS with 70% or 90% EtOH

3. Watch closely under microscope for background to start fading (about 3 min)

4. When desired color is reached, quickly remove EtOH and replace with PBS


4% Paraformaldehyde in PBS

It’s better to use fresh PFA up to 1 week is O.K

Remember to put all leftovers in organic waste

To make 250ml of 4% Paraformaldehyde

Wear disposable gloves throughout the procedure

1.  Use a sterile 250ml Erlenmeyer flask.

2.  Weigh out 10g of paraformaldehyde powder into flask.

3.  Using disposable 50ml centrifuge tube, measure out 40ml of sterile water and put in flask.

4.  Heat to 650c in an H2O bath.

5.  Once solution has warmed to 650c, add 50µl of 10N NaOH, using a P200 tip.

6.  Continue to swirl and heat solution in H2O bath.

7.  Add 25ml of 10X PBS (Phosphate Buffer Saline), and 80µl of 6M HCl.

8.  Bring solution up to 250ml with sterile H2O.

9.  Check pH with litmus paper. Should range from 7.2 to 7.4.

10.  Filter solution with 250ml filter bottle.

11.  Label and place in cold room until ready to use.

PTW: 0.1% Tween-20 in PBS (1X)

Detergent Mix: IGEPAL 500 µl

SDS 5 ml

deoxycholate 0.25 g

1 M Tris-HCl pH8 2.5 ml

500 mM EDTA pH8 100µl

5M NaCl 1.5 ml

DEPC H20 40.4 ml


50.0 ml

Hybridization Mix: Formamide 25.00 ml

SSC (20x pH5 w/citric acid)[3] 3.25 ml

EDTA (0.5 M pH8) 0.50 ml

Yeast RNA[4] (5mg/ml) 0.50 ml {final conc 50 µg/ml}

Tween-20 (10%) 1.00 ml

CHAPS (10%) 2.50 ml

Heparin (50 mg/ml) 0.10 ml {final conc 100 µg/ml}

Water 17.15 ml


Total 50.00 ml

*Store at -20°C

5X MABT: Maleic Acid (50mM) 11.6 g

NaCl (0.74M[5]) 8.7 g

Tween-20 11.0 ml

Water 185.0 ml


Total 200.0 ml

Mix Malex acid in ddw

Adjust pH to 7 using NaOH

Add NaCl (8.7g or 30 ml from 5M solution)

NTMT: 5M NaCl 2.0 ml

1M TrisHCl 10.0 ml

2M MgCl2 2.5 ml

10% Tween-20 10.0 ml

Water 75.5 ml


Total 100.0 ml


NTMT 10 ml

BCIP(50 mg/ml in NTMT) 26.2 µl

NBT (75 mg/ml in DMF*) 33.7 µl

*Dimethyl Formamide

Sheep serum (CHEMICON S22-100mL)

Inactivate 56°c X 30’

Cool on ice

Centrifuge and dived the sup to 1 ml aliquots (important in order to lose the protein precipitate)

BBR (Rochecat. No. 1 096 176)

Prepare X10 stock solution

Dissolve blocking reagent in Maleic acid buffer to final concentration of 10% (w/v)

Mix and heat to 60°c X 1h (do not boil) and make sure reagent is completely dissolved

Store in aliquots of 1.5 ml in –15 to -25°c

Dilute the X10 solution with Maleic acid buffer to a X1 blocking solution on the experiment day. Always prepare fresh

Maleic acid buffer:

100mM Maleic acid

250mM NaCl

adjust pH to 7.5 using conc. Or solid NaOH

[1] Check the phenol color – shouldn’t be brown (EZ-RNA kit has phenol-chloroforme solution)

[2] Our proteinase K is at a 15mg/ml concentration. Add 0.7µl to 1ml of PTW.

[3] 250ml 20SSC pH7 + 50ml 1M citric acid. For a 1M citric acid solution – take 29.4gr in 100ml of nuclease free water.

[4] Other protocols use baker yeast tRNA at a 1mg/ml final concentration.

[5] Take 30ml of a 5M solution.