GUDMAP

Semi-Automated Section in situ Hybridisation for 7µm paraffin sections

This protocol is partially based on protocols by Wilkinson & Nieto Methods Enzymol. 1993;225:361-73 and adapted by members of Group Little and Thierry Gilbert. The Tecan script was supplied by Tecan and Gregor Eichele Dev Dyn. 2005 Oct;234(2):371-86 but has been modified for paraffin sections by Bree Rumballe.

Day 1:  
Dewaxing of slides
Assembly of hybridisation chambers
Pre-hybridisation
Overnight hybridisation with probe
Stringency washes
Blocking
anti-DIG-AP detection  
 
All solutions for Day 1 are made with RO (18ohm) -treated pure water in sterile plastic vials/glassware baked at 180°C.  
 
Day 2:  
Colour development
 
DAY 1:  
DE-WAXING

Ensure RNase-free conditions and solutions at all times.  
Label slides with pencil and assemble into baked steel racks.  
 
In the fume hood, assemble baked glass staining jars and label with appropriate solution name. Make up 300ml of each solution. Take the slides through the following de-waxing process at room temperature:  

1.  Immerse in Xylene 1 for 10 minutes (min).

2.  Immerse in Xylene 2 for 15min.

3.  Rehydrate in 100% Ethanol for 1min.

4.  Rehydrate in 95% Ethanol for 1min.

5.  Rehydrate in 70% Ethanol for 1min.

6.  Wash in PBS for 5min.

7.  Fix in cold 4% paraformaldehyde for 20min.

8.  Wash in PBS for 5min.

9.  Wash in second PBS for 5min.

10.  Transfer to fresh PBS to assemble slides.

Assemble slides into flow-through hybridisation chambers under PBS and ensure no bubbles are present on the slides. Place into degassed chamber racks on the Tecan Freedom Evo platform.  
Ensure all reagents are in the troughs in the correct location on the Tecan platform.  
 
TREATMENT

1.  Begin the Tecan script by incubating slides in Tris buffer (TBS) (0.1M Tris-HCl, 0.15M NaCL, pH7.5) 3 times for 5min at 25°C

2.  Incubate twice for 5min with 0.2N HCl.

3.  Incubate 3 times for 5min with TBS.

4.  Incubate twice for 5-10min with 10ug/ml proteinase K (Roche 3115879) in TBS. Proteinase K is freshly added to the buffer as the script commences, mixed and placed onto the Tecan platform. Adjustment of proteinase K concentration/incubation time may be required for different developmental stages and tissue types. Try 10min for embryonic, 20min for adult tissue.

5.  Incubate once for 5min with PBS.

6.  Fix with 4%paraformaldehyde twice for 5min.

7.  Wash twice for 5min with PBS.

8.  Incubate once for 5min with TBS.

9.  Incubate once for 5min with 0.1M Tris-HCl pH8.0.

10.  Incubate 5 times for 2min with 0.1M triethanolamine and 0.25% (v/v) acetic anhydride (add fresh) in 0.1M Tris-HCl pH8.0.

11.  Incubate once for 5min with 0.1M Tris-HCl pH8.0.

12.  Incubate once for 5min with 2xSSC.

13.  Incubate with hybridisation buffer (50% Formamide, 2xSSC pH5, 1x Denhardt's, 10% Dextran sulphate, 0.2mg/ml tRNA, 0.5mg/ml salmon sperm DNA) at RT at which time the temperature of the chamber rack is increased to 64°C.

At this time the slides may incubate for as long as necessary until the chamber rack is at temperature and the probes are ready, generally between 10-20mins.

HYBRIDISATION

1.  RNA riboprobe (0.3-0.5µg/ml) is mixed with hybridisation buffer, heated at 80°C for 5min, put on ice for 5min, spun and placed on the Tecan platform. A 200µl aliquot is added to each chamber by the Tecan script and 4-5 hours later, a second aliquot of probe is added to the chamber and the hybridisation process is continued for another 4-5hours.

STRINGENCY WASHES

Bottles containing stringency washes are preheated in the incubator at 64ºC and placed into the glass troughs on the Tecan platform. The solutions are maintained at 64°C using a script-controlled circulation bath connected to the chamber.  

1.  Incubate 4 times for 5min with 50% Formamide, 2xSSC (300mM NaCl, 30mM sodium citrate).

2.  Incubate 4 times for 5min with 50% Formamide, 1xSSC.

3.  Incubate 4 times for 5min with 1xSSC. During this time the script ramps down the chamber rack and trough chamber temperature to 25ºC.

4.  Incubate 4 times for 5min with 0.5xSSC.

5.  Incubate 4 times for 5min with 0.2xSSC.

RIBOPROBE DETECTION

1.  Incubate twice for 5min with 1xDIG Wash Buffer (Roche1585762).

2.  Incubate 3 times for 20min with DIG Blocking Buffer (1xDIG Block, 20%heat inactivated sheep serum in 1x malic acid) Roche1585762)

3.  Incubate 3 times for 20min with 1/2000 anti-DIG-AP in DIG Blocking Buffer (Roche11093274910)

4.  Incubate 4 times for 5min with 1x DIG Wash Buffer.

5.  Incubate 4 times for 5min with NTMT (0.1M NaCl, 0.1M Tris-HCl pH9.5, 50mM MgCl2, 0.1% Tween-20).  

Disassemble hybridisation chambers while they are immersed in fresh NTMT, dry the edges and place into NT humidified trays ready for colour development.  

COLOUR DEVELOPMENT  

1.  Mix 200ul pre-mix NBT/BCIP (Roche1681451) per 10ml NT and aliquot 2-300ul per slide or aliquot 200µl of BM Purple (Roche11442074001) directly onto slide. Cover lid with alfoil to create a dark environment and monitor colour reaction.  

2.  Once staining is sufficient, wash with NT for 5min.

3.  Wash with PBS for 5min.  

4.  Fix in 4% parafomaldehyde for 20min.

5.  Wash twice for 5min with PBS.  

6.  Mount and coverslip with aqueous mounting media.

SOLUTIONS for S-ISH using the Tecan Freedom Evo

Xylene
Use RNase-Free (RF) Xylene for both first and second wash. You may re-use the xylene for 4 runs only, provided it has been stored in RF bottles.

100%, 95%, 70% ethanol
300ml are required for each staining jar, use RF 100% ethanol and RO water
95% EtOH = 285ml EtOH + 15ml H2O
70% EtOH = 210ml EtOH + 90ml H2O

10xPBS (phosphate buffered saline) Stock solution
Dissolve 10 RF PBS tablets in 1L RO-H2O. Autoclave
For 1L of 1xPBS solution, use 100ml of 10xPBS + 900ml RO-H2O

16% PFA (paraformaldehyde) Stock solution
Weigh 160g PFA, dissolve in 900ml RF 1x PBS on a magnetic heat stirrer O/N in fumehood
Adjust volume to 1L with RF PBS
Aliquot into 50ml falcon tubes and store at -20°C.
For a 4%PFA solution, add 50ml of 16%PFA to 150ml RF 1xPBS and mix. This may be stored up to 1 week at 4°C.

10xTBS Stock Solution (1.5M NaCl, 0.5M Tris-Cl pH7.5)
For a 1L 10x stock solution, mix 500ml 3M NaCl + 500ml 1M Tris-Cl pH7.5
1x TBS (150 mM NaCl, 50 mM Tris, pH 7.5)
For a 1L 1x solution, add 50ml 3M NaCl,  50ml 1M Tris pH7.5 and 900ml RO-H2O.
Or add 100ml 10xTBS to 900ml RO-H2O

0.2N HCl
0.8 ml HCl 37% in 50 ml 1x TBS. Make this solution fresh each time

Proteinase K Roche3115879 (10 µg/ml) in TBS
Use 25µl of a 20mg/ml stock solution (stored in aliquots at -20°C and mix into 50ml 1xTBS. Make fresh for each run. Proteinase K should be tested for each new batch.

0.1M Tris pH 8.0
Add 100ml 1MTris pH8.0 to 900ml RO H2O

Acetylation Solution
Add 1ml TEA and 0.250ml acetic anhydride per 100ml of 0.1M Tris pH8.0. Make up solution and just before adding to slides, add the acetic anhydride.

20xSSC stock, pH7
Dissolve 175.3gNaCl and 88.2g sodium citrate-2H2O in 800ml H2O.
Adjust pH to 7.0 with HCl and adjust vol to 1L with water.
Autoclave and store at RT.
For 200ml solutions,
2xSSC  20ml 20xSSC, 180ml water
1xSSC  10ml 20xSSC, 190ml water
0.5xSSC 5ml 20xSSC, 195ml water
0.2xSSC 2ml 20xSSC, 198ml water

Hybridisation Buffer
For 50ml, 
25ml  50% Formamide
5ml 20xSSC 2x SSC, pH5
1ml 50x  1x Denhardt
10ml  50% 10% Dextran sulfate
1ml 50x  0.2mg/ml tRNA
1ml 50x  0.5mg/ml ssDNA
Xml  RO-H2O to make up to 50ml total volume

50% dextran sulphate stock
Dissolve 50g dextran sulphate in 100ml RO-H2O. Add powder a little at a time to the water on a heated magnetic stirrer to avoid clumping. Once dissolved, aliquot and store at -20°C.

50x tRNA stock
Dissolve 200mg in 20ml RO-H2O and rotate in hyb oven at 55-65degC O/N.
Aliquot into 1ml tubes and store at -20°C.

50x salmon sperm DNA stock
Mix 500mg in 10ml RO-H2O, shear with a 17 gauge needle and adjust volume to 20ml.
Aliquot into 1ml tubes and store at -20°C.

Stringency Wash Buffer 1 (50%F, 2xSSC)
For 500ml, 250ml formamide + 50ml 20xSSC + 200ml RO-H2O

Stringency Wash Buffer 2 (50%F, 1xSSC)
For 500ml, 250ml formamide + 25ml 20xSSC+ 225ml RO-H2O

1xRoche DIG Wash Buffer DIG Wash Block & Buffer Set Roche1585762
For 100ml, use 10ml 10x stock + 90ml RO-H2O

Roche DIG Blocking buffer
For 100ml, add
10ml 10x Roche Malic Acid
10ml 10x Roche Block
20ml heat inactivated sheep serum
60ml RO-H2O

Fab anti-DIG (Roche11093274910) in Blocking Buffer, 1%Tween20
For 50ml, add 1/2000 = 25ul anti-DIG-Ab to 50ml Blocking Buffer and 0.5ml 10% Tween20

NT (0.1M NaCl, 0.1M Tris pH9.6)   
For 1L of 1xNT
33.33ml 3M NaCl 
100ml 1M Tris pH9.6

NTMT (0.1M NaCl, 0.1M Tris pH9.6, 50mM MgCl2, 0.1% Tween20 w/v)
For 100mL of 1xNTMT,
5ml 1M MgCl2
1ml 10% Tween20
3.33ml 3M NaCl
10ml 1M Tris pH9.6
80.67ml RO-H2O

NBT/BCIP solution Roche1681451 RT, dark
Use 20ul pre-mix NBT/BCIP Stock solution (Roche1681451) per 1ml NT

BM Purple, Roche11442074001 RT, dark
Use undiluted BM Purple and directly aliquot onto slides