TITLE: Fluorescent in situ hybridisation for juvenile Fasciola hepatica
AUTHORS: Duncan Wells, Paul McCusker, Rebecca Armstrong, Emily Robb

[DESCRIPTION]
This FISH protocol has been adapted for use in Fasciola hepatica, specifically 4-week-old juveniles, grown in vitro in 50% Chicken Serum, 50% RPMI culture medium. The majority of this protocol comes from those of the Newmark Lab (King and Newmark, 2013) and Collins Lab (Collins and Collins, 2017). We are grateful to members of these labs for several helpful discussions on this technique.

[BEFORE_START]
Ensure in situ oven is at the desired temperature. We usually switch the oven on at the start of the day 1 and allow the oven to reach a stable temperature.

[GUIDELINES]
Following a long period of relative success with this technique, we had noticed difficulties emerged. After trying various tweaks, we decided to purchase the vast majority of reagents again. This seemed to have helped significantly.

For all steps prior to and including the riboprobe incubation, all tips and tubes should be RNase free. Reagents should be made up with DEPC-treated double distilled H2O - for 1 litre of DEPC-treated water, add 1ul Diethyl pyrocarbonate (D5758, Sigma) per 1ml of H2O. Shake vigorously for 15 seconds. Leave at room temperature overnight, or 2 hours at 37oC, then autoclave.

We have found that longer riboprobes (800-1000 bases) have generally worked better than shorter (150-300 bases) ones.

Alongside every positive test, we run the sense probe as a negative control. This is especially helpful when we see probes with otherwise ambiguous/questionable staining.

Highlighted solutions have recipes at the bottom of the protocol.

[STEPS]
SECTION: Worm Preparation
19. Carry out excystment (see McVeigh et al., 2014), transfer newly excysted juveniles to 96-well round-bottomed plates, in groups of <25 NEJs per well. Add 200μl of pre-warmed, pre-mixed 50:50 chicken serum (New Zealand Origin, 16110082, Thermo) and RPMI (11835030, Thermo). Replace this every other day. Worms are maintained in a 37 °C incubator with 5% CO2.

SECTION: Worm Preparation
20. At 4 weeks old, wash juveniles out of 50:50 and add RPMI. Transfer worms into a small petri dish (Starstedt – 83.3925) containing 0.25% Tricaine diluted in RPMI. Leave worms in this for 3 min. 
 


SECTION: Worm Preparation
21. Transfer worms in a minimal amount of tricaine/RPMI into a 20μl droplet of 4% Formaldehyde and set a glass coverslip on top. Leave for 10 min. Remove the coverslip and collect worms into 1.5ml hydrophobic tubes (1210-10, SSIBio). Add 1ml 4% formaldehyde. Rotate tubes for 10 min.

SECTION: Worm Preparation
22. Remove 4% formaldehyde and wash once with PBST, then replace this with a pre-mixed 1:1 ratio of PBST and methanol (32213, Sigma) rotating for 10 min. Worms can then be stored in 100% methanol at -20 °C until use. We are yet to test how long worms can be stored for and still be suitable. We would be reluctant to use worms stored for longer than 6 months.

SECTION: Riboprobe Preparation
1. Amplify target of interest with an end-point PCR using FastStart Taq polymerase, dNTPack (4738381001, Sigma). Primers should have a 5’-TAATACGACTCACTATAGGGT-3’ promoter sequence. For the antisense probe (positive) use the regular forward primer and the T7-tagged reverse primer. For the sense probe (negative control) use the T7-tagged forward and the regular reverse.

SECTION: Riboprobe Preparation
2. Purify resulting PCR products with the ChargeSwitch PCR clean-up kit (CS12000, Thermo) according to the manufacturers guidelines. Only 15μl of elution buffer is used to maximise amplified target concentration.
SECTION: Riboprobe Preparation
3. Perform a transcription reaction containing 2μl transcription buffer, 1μl T7 polymerase (both from T7 polymerase kit, EP01111), 6μl purified PCR product, 1μl DIG-RNA labelling mix (11277073910, Sigma). Incubate    at 27 °C.

SECTION: Riboprobe Preparation
4. DNase treat the probes by adding 0.25μl DNase I (18047019, Thermo), 0.25μl transcription buffer (from previous step) and 2μl RNase-free H2O. Incubate at 37 °C for 15 min.

SECTION: Riboprobe Preparation
5. Add 1.25μl 4M LiCl and 37.6μl molecular-grade ethanol (51976, Sigma). Precipitate probes    at -80 °C.

SECTION: Riboprobe Preparation
7. Remove supernatant, wash pellet with 70% ethanol, 16000 x g, 5 min, 4 °C , discard supernatant and allow to air-dry for 15 min.
 

SECTION: Riboprobe Preparation
8. Resuspend pellet in 20μl RNase-free H2O. Check purity and concentration on a spectrophotometer/fluorometer (we use a DeNovix DS-11 FX).
SECTION: Riboprobe Preparation
9. Adjust concentration to 50ng/μl with Hybridisation Buffer, store probes at -20 °C until use.

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
10. Add 1g of torula RNA (RNA from torula yeast, type VI [R6625, Sigma]) to a 50ml conical tube.

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
11. Add 10ml phenol (P4557, Sigma) and 15ml Tris-EDTA buffer (12090015, Thermo). Vortex tubes until the yeast RNA has dissolved. 16000 x g, 10 min, 4 °C 

SECTION: Riboprobe Preparation
6. 16000 x g, 20 min, 4 °C 

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
12. Collect the aqueous phase into a new tube (50ml), add 1:1 phenol/chloroform (P2069, Sigma), mix well, 16000 x g, 10 min, 4 °C .

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
13. Collect aqueous phase into a new tube, add 1:1 chloroform, mix well, 16000 x g, 10 min, 4 °C 

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
14. Collect aqueous phase into a new 50ml tube, add 1ml 3M sodium acetate (71196, Sigma) and 30ml molecular-grade ethanol. Leave at -20 °C    to precipitate.

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
15. 16000 x g, 20 min, 4 °C , discard supernatant, wash pellet in 70% ethanol, 16000 x g, 10 min, 4 °C 

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
16. Remove supernatant, allow pellet to airdry for 15 min, add 5ml formamide pre-heated to 50 °C

SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
17. Vortex until the pellet is resuspended
SECTION: Preparation of yeast RNA (mainly from Jiang [2012])
18. Measure concentration and purity on a spectrophotometer/fluorometer. Adjust concentration to 50mg/ml and store in 1ml aliquots at -20 °C.

SECTION: Recipes (presented in order of first appearance) (DEPC-treated H2O presented as DT-H2O, double distilled water presented as ddH2O)
49. 4M LiCl (100ml)
Add 16.96g LiCl (L9650, Sigma) to 50ml DT-H2O. Add this slowly as this is an exothermic reaction. Once dissolved, bring volume to 100ml with ddH2O. 

Hybridisation Buffer (50ml)
25ml deionised formamide (F9037, Sigma)
12.5ml 20xSSC (S6639, Sigma)
100μl 1mg/ml yeast RNA
5ml 10% (v/v) Tween-20 (5ml Tween-20 [P1379, Sigma] + 45ml DT-H2O)
5ml 50% Dextran sulfate (S4030, Sigma)
2.4ml DT-H2O

0.25% Tricaine
50mg Ethyl 3-aminobenzoate methanesulfonate (E10521, Sigma) in 2ml RPMI

4% Formaldehyde (4.5ml)
500μl 36.5% formaldehyde (F8776, Sigma)
4ml PBST (see below)

PBST (200ml)
1 tab PBS (P4417, Sigma)
200ml DT-H2O
600μl Triton-X (T8787, Sigma)

1xSSC (20ml)
1ml 20xSSC
19ml DT-H2O

Bleaching solution (2ml)
1.77ml DT-H2O
100μl deionised formamide
50μl 20xSSC
80μl H2O2 (H1009, Sigma)

Proteinase K solution (~5ml)
4.95ml PBST
50μl 10% SDS (dissolve 5g SDS [L4509, Sigma] in 50ml DT-H2O)
2.5μl Proteinase K (03115887001, Sigma) (check the bottle for exact stock concentration as this varies, adjust calculation accordingly. End concentration is to be 10ug/ml)

Prehybridisation buffer (50ml)
25ml deionised formamide
12.5ml 20xSSC
100μl 50mg/ml yeast
5ml 10% (v/v) Tween-20
7.4ml DT-H2O

2xSSC (20ml)
17.98ml ddH2O
2ml 20xSSC
20μl Triton-X

0.2xSSC (20ml)
19.78ml ddH2O
200ul 20xSSC
20μl Triton-X

TNT (~1L)
1L ddH2O
12.11g Tris Base (T1503, Sigma)
8.77g NaCl (27800.291, VWR)
3ml Triton-X
pH to 7.5 and filter sterilise

Blocking Solution (8ml)
400μl Horse serum (H0146)
40μl Western Blocking Solution (11921673001, Sigma)
7.56ml TNT

Antibody solution (4ml)
2μl Anti-DIG-AP (Chromogenic ISH) (11093274910, Sigma) / 3ul Anti-DIG-HRP (Fluorescent ISH) (11207733910, Sigma)
3998μl Blocking Solution

Tyramide Solution (~5ml)
5ml TSA Buffer (see below)
1μl 20mg/ml 4-iodophenylboronic acid (see below)
6μl 5% H2O2 (492μl TSA Buffer + 8μl H2O2)
2μl FAM-Tyramide (see below)

TSA Buffer (200ml)
58.44g NaCl 
3.09g Boric acid (B6768, Sigma)
100ml ddH2O, bring to 200ml when dissolved
pH to 8.5 and filter sterilise

20mg/ml 4-iodophenylboronic acid (2ml)
40mg 4-iodophenylboronic acid (IPBA, 471933, Sigma) in 2ml dimethylformamide (DMF, 227056, Sigma)

FAM-Tyramide
 - Tyramine solution = Dissolve 10mg tyramine hydrochloride (T2879, Sigma) in 1ml DMF, add 10μl triethylamine (T0886)
 - FAM-DMF = Dissolve 10mg 5(6)-FAM, SE (5-(and-6)-Carboxyfluorescein, Succinimidyl Ester) (C1311, Thermo) in 1ml DMF
Add 342.5μl tyramine solution to 1ml FAM-DMF
Incubate at for 120 min, rotating at room temperature
Add 8.6ml ethanol for a 1mg/ml stock
Split into 20μl aliquots and store at -20 °C 

Exposure Buffer (~10ml)
Prepare 50ml tubes of each of;
 - 1M Tris base = 6.06g
 - 1M NaCl = 2.92g
 - 1M MgCl2 = 4.76g
Bring each to 50ml with ddH2O

Prepare PVA - bring 80ml ddH2O to 75 °C  while stirring and slowly add 10g PVA (P8136, Sigma). If you add this too quickly it will form clumps and not dissolve.

1ml 1M Tris base
1ml 1M NaCl
500μl 1M MgCl2
100μl 10% Tween-20
7.4ml PVA
45μl 4-Nitro blue tetrazolium (NBT, 11383213001, Sigma)
35μl 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, 11383221001, Sigma)
	
DAPI Solution (10ml)
10μl 1mg/ml DAPI (dissolved in ddH2O) (D9542, Sigma)
9.99ml TNT
SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
23. Remove 100% methanol from worms and replace with 1:1 PBST:Methanol for 10 min rotating.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
24. Add PBST, incubate for 10 min rotating

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
26. Incubate worms in 200μl Bleaching solution under a bright light for 60 min

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
27. Add 1xSSC and incubate for 10 min with no rotation

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
28. Add PBST, incubate for 10 min rotating.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
29. Add Proteinase K solution, incubate for 15 min with no rotation.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
30. Add 4% formaldehyde, incubate for 10 min rotating.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
31. Add PBST

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
32. Transfer worms in 500μl PBST into in situ baskets (12.444, Intavis) in a 24-well plate. Add 500μl Prehybridisation buffer.
SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
33. Add plate to a shaker for 10 min set at 100rpm (any future shaking steps are at 100rpm)

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
34. Add Prehybridisation buffer (pre-warmed to 52 °C) to the next well and transfer the baskets to these. Place the plate in an in situ oven pre-warmed to 52 °C. Have the plate rocking. Our in situ oven (Boekel Scientific Shake 'N Bake™ Rocking Hybridization Oven/Incubator, 136400) only allows for gentle rocking.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
35. Add the probe mix to the next well, transfer baskets to this well and incubate   rocking at 52 °C.

SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
34.1. Approximately 10 minutes before the end of the above 2 hours, prepare the probe as follows - heat 20μl of 50ng/μl probe to 80 °C for 5 min then place on ice. Add 980μl Hybridisation buffer to the probe.

SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
36. Remove 500μl of probe mix from the well with the worms and add 500μl pre-heated 2xSSC, incubate for 20 min rocking at 52 °C .

SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
37. Wash in 2xSSC, incubate for 20 min rocking at 52 °C .

SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
38. Repeat step 36) 2 more times, moving baskets into new wells each time.
SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
39. Wash with 0.2xSSC, incubate for 20 min rocking at 52 °C 

SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
40. Repeat step 38) 3 more times, moving baskets into new wells each time.
SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
41. Wash with TNT, incubate for 10 min shaking at Room temperature  
SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
42. Repeat step 41) once
SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
43. Incubate in Blocking solution for 60 min shaking at Room temperature 
SECTION: Day 2 FISH (volumes are 1ml unless otherwise stated) (each basket wash is carried out in a new well unless otherwise stated)
44. Incubate in Antibody solution 60 min shaking at 4 °C 

SECTION: Day 3 FISH (volumes are 1ml unless otherwise stated)
45. Wash with TNT for 5 min , then 10 min , and then 6x20 min shaking at Room temperature .
SECTION: Day 3 FISH (volumes are 1ml unless otherwise stated)
46. Protect plates from light from this point on. 

FOR FLUORESCENT ISH
Incubate in freshly-made Tyramide solution for 10 min , shaking at Room temperature . Go to step 46).

FOR CHROMOGENIC ISH
Transfer worms to small petri dishes and incubate in Exposure buffer for up to 240 min  with no rotation, checking every 20 minutes or so. If no signal has developed after 4 hours, incubate the dishes at 4 °C . This slows any subsequent development but this allows you to leave it overnight. When a sufficient signal has developed, halt the reaction by washing worms with TNT. Wash the worms in 100% ethanol to reduce any background/non-specific staining. Wash worms with TNT. Go to step 47).

SECTION: Day 3 FISH (volumes are 1ml unless otherwise stated)
47. Incubate worms in DAPI solution for 20 min, followed by two 10 minute washes in TNT.

SECTION: Day 3 FISH (volumes are 1ml unless otherwise stated)
48. Mount worms in 10μl Vectashield (H-1000, Vector Labs) on standard microscope slides and seal coverslips with clear nail polish.
SECTION: Day 1 FISH (each step begins with removing the solution from the last) (volumes are 1ml unless otherwise stated)
25. Add 1xSSC and incubate for 10 min with no rotation