Edinburgh Core Database Group

Capel Group

Cohn Group

Gaido Group

Jain Group

Keast Group

Lessard Group

Little Group

McMahon Group

Mendelsohn Group

Potter Group

Southard-Smith Group

Vezina Group

 

 

Little Group Protocols

Semi-Automated Whole Mount in situ Hybridisation

Using the BioLane HTI Robot

The following method is partially based on the protocol by Wilkinson & Nieto Methods Enzymol. 1993;225:361-73 and adapted by Gemma Martinez and Brooke Gardiner.

BioLane HTI Robot

 

The BioLane HTI robot consists of two systems, a blue and a red system.  Each system has 9 plastic tubes hooked into the robot labeled 1 to 9. The robot has been programmed so each tube has been assigned to a specific solution. By inserting the tube into the solution the robot will automatically take the required amount as generated by a script. The robot will gently rock the samples in a tray while the program is in progress.

Nylon mesh baskets are used by the robot to hold the tissues, each basket represents one probe. Three sizes tray are available with the BioLane HTI robot, each holds a different number of nylon mesh baskets. We routinely use the small (20 basket) tray.

Pretreatment of Embryos

1. Dissect embryos of 9.5dpc (whole embryo), 10.5dpc (forelimb-hindlimb) and 12.5dpc(urogenital tract) in ice-cold PBS.

2. Immediately fix the tissues with 4% paraformaldehyde (PFA) overnight (16-18hours) at 4°C.

3. Wash the tissues twice with PBTX for 10 minutes each at room temperature.

4. Dehydrate with a series of 25% MeOH/75%PBTX, 50%MeOH/50%PBTX, 75% MeOH/25%PBTX washes for 20 minutes each, follow by two washes of 100% MeOH for 20minutes.

5. Store embryo tissues in 100%MeOH at -20°C.

Day 1- Cleaning, Re-hydration and Hybridisation (Blue System)

1.  Place tray with nylon mesh baskets into the blue system and run the cleaning program.

2. Wash all the tubing in the blue system with 0.2M NaOH. (This is to maintain an RNase-free environment)

3. Wash all the tubing in the blue system with RO-H2O.

4. Carefully put the embryonic tissues into the baskets. Each basket contains 2x 9.5dpc, 3x 10.5dpc, 1 male and 1 female 12.5dpc, and 2 x12.5dpc/2d explants.

5. Start the re-hydration program of the blue system.

6.  The samples are treated as follows:

Re-hydration

RT

 5 Minutes

100% MeOH

 

RT

 5 Minutes

75% MeOH/ 25% PBTX

 

RT

 5 Minutes

50% MeOH/ 50% PBTX

 

RT

 5 Minutes

25% MeOH/ 75% PBTX

 

RT

10 Minutes

PBTX

 

RT

 5 Minutes

PBT

 

RT

60 Minutes

PBT/ 6% H2O2

 

RT

 5 Minutes

PBT

 

RT

 5 Minutes

PBTX

 

RT

 5 Minutes

PBTX

Digestion

RT

20 Minutes

10ug/ml Proteinase K/ PBTX

 

RT

 5 Minutes

PBTX

 

RT

 5 Minutes

PBTX

7. Turn off robot and wash samples with 0.2% gluteraldehyde/4% PFA in PBTX in the fume hood at room temperature for 20 minutes.

8. Wash twice with PBTX at room temperature for 10 minutes each.

9.  Remove baskets from tray and place baskets into a 48 well Cellstar plate containing 0.5ml pre-hybridisation solution in each well at 65°C for 2 hours.

10.  Transfer baskets to a 48 well Cellstar plate with 0.5ml hybridisation solution at 65°C overnight. (Hybridisation solution = pre-hybridisation solution + ~0.2µg/ml DIG-labelled RNA probe.)

Preparation

11. Prepare the post-hybridisation washes and place at 65°C overnight.

Preabsorption of anti-DIG antibody

12. Prior to starting post-hybridisation washing, prepare the pre-blocking solution and pre-absorb the anti-DIG antibody Roche11093274910. (The pre-absorbed anti-DIG antibody can be kept at 4°C and recycled up to 3 times.)

18mg of embryo powder is placed in a 10ml tube with 10% sheep serum, 2% BSA in TBTX and 15µl anti-DIG antibody.

Incubate at 4°C for 3 hours or longer with gentle rocking.

Spin sample in a microfuge for 10 minutes, 4°C at 13000rpm.

Collect the supernatant and dilute it with 10% sheep serum, 2% BSA in TBTX.

Store the antibody at 4°C until use.

DAY 2 – Post-Hybridisation and pre-blocking (Red system)

1. Remove the baskets from the 48 well plate and place in a tray filled with solution 1.

2. The robot will preheat the rocker with the samples to 65°C for 15 minutes.

3. The following stringency washes are performed:

Post-Hybridisation

65°C     

 5 Minutes

100% Solution 1

washes

65°C

 5 Minutes

75% Solution1/25% 2x SSC

 

65°C

 5 Minutes

50% Solution 1/50% 2x SSC

 

65°C

 5 Minutes

25% Solution 1/75% 2x SSC

 

65°C

10 Minutes

2x SSC / 0.1% CHAPS

 

65°C

10 Minutes

2x SSC / 0.1% CHAPS

 

65°C

 5 Minutes

0.2x SSC / 0.1% CHAPS

 

65°C

5 Minutes

0.2x SSC / 0.1% CHAPS

 

RT     

10 Minutes

TBTX

 

RT     

10 Minutes

TBTX

Pre-blocking

RT      

2 Hours

Pre-Block Solution

 

4°C             

Overnight

Pre-absorb anti-DIG antibody

Preparation

4. Prepare 0.1% BSA / TBTX at the end of day 2 and insert tubing into solution. The robot will start taking in 0.1% BSA / TBTX early morning of day 3.

Day 3 – Post-antibody washes and Development

1. The samples are washed as follows:

Post-Antibody

RT

30 Minutes

0.1% BSA / TBTX

washes

RT

30 Minutes

0.1% BSA / TBTX

 

RT

30 Minutes

0.1% BSA / TBTX

 

RT

30 Minutes

0.1% BSA / TBTX

 

RT

30 Minutes

0.1% BSA / TBTX

 

RT

10 Minutes

TBTX

The pH for NTMT decreases over time so it needs to be made fresh.  The robot will pause for you to make up fresh NTMT and re-start it

 

RT

10 Minutes

TBTX

 

RT

10 Minutes

NTMT

 

RT

10 Minutes

NTMT

 

RT

10 Minutes

NTMT

2.  Transfer contents of the baskets into 12 well plates with 1ml of BM purple substrate Roche11442074001 in each well.

Bring the BM purple AP substrate to 15°C –20°C before using, no dilution is required but you may need to filter the solution to remove any precipitate.

3.  Wrap the 12 well plates with aluminium foil and monitor samples for colour development approximately every 15-30 minutes.

Note: Normally 3 control samples will be used to verify the success of the run. Controls are Wnt4 (strong), Wnt7b (medium) and Shh (weak). For Wnt4 and Wnt7b, generally the expression develops within 1 to1.5hours whereas Shh can take 25-30 hours.

4.  When the colour has proceeded to the desired extent, stop the colour development by transferring the samples to a new 12 well plate containing distilled water. Depending on the extent of colour development, samples can be left overnight to continue developing or kept at 4°C to slow down the reaction.

Note: If the samples have been over-developed, wash several times in PBS with 1% Triton X-100. This can be done for several days if necessary and will decrease the background issue.

4. Wash 3x with PBS at room temperature for 5 minutes each.

5. Fix samples with cold 4% paraformaldehyde at room temperature for 30 minutes.

6. Wash 3x with PBS at room temperature for 5 minutes each.

7. Store samples in 2ml Eppendorf tubes with PBS at 4°C.

8. Photograph as soon as possible. Use a petri dish with 1% agarose as a background.


Solutions & Volumes Required For W-ISH Using The Biolane Hti Robot

 Small Tray (20 Baskets)

DAY1

Methanol         70mL

PBTX                350mL

PBT            80mL

PBT/H2O2             50mL

Proteinase K          50mL

Glut/PFA/PBTX       40mL

PBT/H2O2

10mL  H2O2

40mL  PBT

Proteinase K (Roche3115879)

25uL  20mg/mL stock proteinase K

50mL  PBTX

Glut/PFA/PBTX

320uL  25% glutaraldehyde

48uL    Triton X-100

40mL   4% PFA in PBS

DAY2

Pre-Block         50mL

Antibody           50mL

Solution1         70mL

2xSSC              70mL

2xSSC+CHAPS 80mL

0.2XSSC+CHAPS     80mL

TBTX                180mL

TBTX+0.1%BSA  200mL

NTMT               120mL

Pre-Block (100mL) N.B. If re-using antibody only require 50mL

2g BSA

10mL Sheep Serum

90mL TBTX

Antibody – Can store in fridge and re-use up to 3 times

15uL  anti-DIG antibody (Roche11093274910)

3mL   Pre-Block

18mg Embryo powder

Incubate this solution on rocker in cold room for 3 hours minimum and then spin down at 13 000 rpm for 10min. Collect the supernatant and add to 50mL pre-block solution before using in the in situ hybridisation.

Solution 1

50mL Formamide

25mL 20x SSC

100uL Triton X-100

10mL 5% CHAPS

15mL water

2xSSC+CHAPS (80mL)         2xSSC+CHAPS (100mL)

8mL 20xSSC                            10mL 20xSSC

1.6mL 5% CHAPS              2mL 5% CHAPS

70.4mL H2O                       88mL H2O

0.2xSSC+CHAPS (80mL) 0.2xSSC+CHAPS (100mL)

0.8mL 20x SSC                  1mL 20x SSC

1.6mL 5% CHAPS              2mL 5% CHAPS

77.6mL H2O                       97mL H2O

TBTX+0.1%BSA

0.2g BSA

200mL TBTX

NTMT

12mL  1M NaCl

12mL  1M Tris.Cl pH9.5

6mL    1M MgCl2

120uL  Tween 20

90mL   H2O

Colour development solution

BM Purple (Roche 11442074001)

Solution Preparation for the W-ISH

PBTX

   

Volume 

Component

Final Concentration

50µl

Triton X-100

0.10%

50ml

1x PBS

 

50ml

Total volume

 
     

TBTX

   

Volume 

Component

Final Concentration

12.5ml

1M Tris.HCl pH 7.5

50mM

37.5ml

1M NaCl

150mM

250µl

Triton X-100

0.10%

199.75ml

Nuclease free H2O

 

250ml

Total volume

 
     

NTMT (prepare fresh when needed-pH decrease over time)

Volume 

Component

Final Concentration

5ml

1M NaCl

100mM

5ml

1M Tris HCl pH9.5

100mM

2.5ml

1M MgCl2

50mM

50µl

100% Tween 20

0.10%

37.45ml

Nuclease free H2O

 

50ml

Total volume

 
     

Solution 1

   

Volume 

Component

Final Concentration

25ml

Formamide

50%

12.5ml

20x SSC pH5

5x

50µl

Triton X-100

0.10%

5ml

CHAPS

0.50%

7.45ml

Nuclease free H2O

 

50ml

Total volume

 

2x SSC/ 0.1% CHAPS

   

Volume 

Component

Final Concentration

 

5ml

20x SSC pH5

2x

 

1ml

5% CHAPS

0.10%

 

44ml

Nuclease free H2O

   

50ml

Total volume

   

0.2x SSC/ 0.1% CHAPS

 

Volume 

Component

Final Concentration

0.5ml

20x SSC pH5

0.2x

1ml

5% CHAPS

0.10%

48.5ml

Nuclease free H2O

 

50ml

Total volume

 
     

in situ pre-block

   

Volume 

Component

Final Concentration

1ml

100% Sheep serum

10%

0.2g

BSA powder

2%

~8.8ml

TBTX

 

10ml

Total volume

 
     

PBT

   

Volume 

Component

Final Concentration

2ml

Tween 20

1%

200ml

PBS

 
     

5% CHAPS

   

Volume 

Component

Final Concentration

2.5g

CHAPS

5%

50ml

Nuclease free H2O

 
     

4% Paraformaldehyde

 

Volume 

Component

Final Concentration

2g

Paraformaldehyde

4%

50ml

1x PBS

 

 

20x SSC

   

Volume 

Component

Final Concentration

87.65g

NaCl

3M

44.1g

Na Citrate

0.3M

to 400ml

Nuclease free H2O

 

pH to .0

10M NaOH

 

to total

Nuclease free H2O

 

500ml

Total volume

 
     

Pre-Hybridisation Solution

 

Volume 

Component

Final Concentration

50ml

Formamide

50%

25ml

20x SSC

5x

2g

Blocking powder

2%

100µl

Triton X-100

0.10%

10ml

5% CHAPS

0.50%

100mg

Torula Yeast RNA

1mg/ml

1ml

0.5M EDTA

5mM

5mg

Heparin

50ug/ml

to total

Nuclease free H2O

 

100ml

Total volume

 
     

* Torula Yeast RNA can be added straight to the solution

     

1M EDTA

   

Volume 

Component

Final Concentration

93.05g

EDTA

1M

to 200ml

Nuclease free H2O

 

pH to 8

10M NaOH

 

to total

Nuclease free H2O

 

250ml

Total volume

 

 

0.5M EDTA

   

Volume 

Component

Final Concentration

93.05g

EDTA

0.5M

to 400ml

Nuclease free H2O

 

pH to 8

10M NaOH

 

to total

Nuclease free H2O

 

500ml

Total volume

 
     

1M Tris

   

Volume 

Component

Final Concentration

121.1g

Tris-base

1M

to 800ml

Nuclease free H2O

 

pH

Concentrated HCl

 

to total

Nuclease free H2O

 

1000ml

Total volume

 

* Approximate Volumes for pH correction for 1000ml Tris

70ml

Concentrated HCl

pH 7.4

60ml

Concentrated HCl

pH 7.6

42ml

Concentrated HCl

pH 8.0

     

If the 1M solution has a yellow colour, discard it and obtain better quality Tris

     

Mouse Embryo Powder

 

~12.5 - 14.5 dpc mouse embryos homogenized in minimal volume PBS

+ 4x volume acetone (0oC)

 

0oC at 30 minutes

   

Spin 10 000g 10 minutes

 

Remove and discard supernatant

 

+ acetone (0oC)

   

Spin 10 000g 10 minutes

 

Remove and discard supernatant

 

Spread pellet out and grind to fine powder on a sheet of filter paper

Allow to air dry

   

4 oC at storage in airtight container

 

 


Group Description