GUDMAP

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			10%
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