TITLE: EdU Staining Protocol in juvenile F. hepatica
AUTHORS: Paul McCusker, Duncan Wells, Rebecca Armstrong, Emily Robb, Paul McVeigh, Nathan Clarke, Erica Gardiner, Erin McCammick, Aaron Maule

[DESCRIPTION]
This protocol describes the process of labelling and whole mount staining proliferative cells in in vitro juvenile Fasciola hepatica with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU). The protocol is based on the Schistosoma mansoni EdU staining protocol developed by the Collins and Newmark groups. A full copy of that protocol is available here - http://collinslab.org/PDF/Whole_mount_EdU_Smansoni.pdf.

[GUIDELINES]
Fixation
While flat fixing is necessary for worms grown for more than three weeks in vitro younger worms may be suitable for free fixing in 4%FA.

[STEPS]
SECTION: In vitro addition of EdU to juvenile F. hepatica
1. Juvenile liver fluke are cultured in 50% Chicken serum (CS50) as described in McCusker et al. (2016).
2. 10 µL of 10 millimolar (mM) 5-Ethynyl-2'-deoxyuridine (EdU) is mixed with 190 µL pre-warmed CS50 and added to worms for between 6 and 24 h.
SECTION: Wash out EdU and chase
3. At conclusion remove EdU from worms 

SECTION: Wash out EdU and chase
4. Wash worms x3 in 100 µL pre-warmed RPMI.
SECTION: OPTIONAL Storage Step
9. Wash in 1 mL  PBSTx for 10 min.

SECTION: OPTIONAL Storage Step
10. Wash in 1 mL  1:1 PBSTx:Methanol for 10 min.

SECTION: OPTIONAL Storage Step
11. Store in 1 mL  methanol at -20 °C until required.

SECTION: OPTIONAL Storage Step
12. Rehydrate in 1 mL 50:50 PBSTx:Methanol for 10 min.

SECTION: Staining
13. x2 washes in 1 mL PBSTx for 10 min each.



SECTION: Staining
14. Incubate in 1 mLfresh Proteinase K Solution for 15 min at Room temperature .

SECTION: Staining
15. Postfix in 1 mL4%FA for 10 min.

SECTION: Staining
16. Wash in 1 mL PBSTx for 10 min.
SECTION: Staining
17. Incubate in EdU Detection Solution for 30 min at Room temperature . NB: cover with tinfoil from this point on.

SECTION: Staining
18. x2 washes in 1 mL PBSTx for 5 min each
SECTION: Staining
19. Incubate in 1 mL DAPI Solution for 20 min at Room temperature or O/N at 4 °C .

SECTION: Staining
20. Rinse in PBSTx and mount in  .

SECTION: Fixation
6. Add 20 µL 4% FA/4%PFA to a petri dish. 
SECTION: Fixation
7. Pipette worms in into 4% FA/4%PFA and immediately flatten with coverslip and weight (e.g. half full 15 mL falcon tube) for 10 min.
SECTION: Fixation
8. Add 4% FA/4%PFA to float coverslip. Lift coverslip and move worms into 1.5 mL eppendorf with 1 mL 4% FA/4%PFA. Place tube on a rotator for further 10 min (4%FA)/2 h (4%PFA) at Room temperature  OR O/N at4 °C. 

SECTION: Wash out EdU and chase
5. Add fresh CS50 to worms and return to culture until desired timepoint.
SECTION: Fixation
3. Add 20 µL 4% FA/4% PFA to a petri dish. 
SECTION: Fixation
4. Pipette worms in into 4% FA/4% PFA and immediately flatten with coverslip and weight (e.g. half full 15 mL falcon tube) for 10 min.
SECTION: Fixation
5. Add 4% FA/4% PFA to float coverslip. Lift coverslip and move worms into 1.5 mL eppendorf with 1 mL 4% FA/4% PFA. Place tube on a rotator for further 10 min (4%FA)/2 h (4%PFA) at Room temperature  OR O/N at4 °C. 

SECTION: OPTIONAL Storage Step
6. Wash in 1 mL  PBSTx for 10 min.

SECTION: OPTIONAL Storage Step
7. Wash in 1 mL  1:1 PBSTx:Methanol for 10 min.

SECTION: OPTIONAL Storage Step
8. Store in 1 mL  methanol at -20 °C until required.

SECTION: OPTIONAL Storage Step
9. Rehydrate in 1 mL 50:50 PBSTx:Methanol for 10 min.

SECTION: Staining
10. x2 washes in 1 mL PBSTx for 10 min each.



SECTION: Staining
11. Incubate in 1 mLfresh Proteinase K Solution for 15 min at Room temperature .

SECTION: Staining
12. Postfix in 1 mL4%FA for 10 min.

SECTION: Staining
13. Wash in 1 mL PBSTx for 10 min.
SECTION: Staining
14. Incubate in EdU Detection Solution for 30 min at Room temperature . NB: cover with tinfoil from this point on.

SECTION: Staining
15. x2 washes in 1 mL PBSTx for 5 min each
SECTION: Staining
16. Incubate in 1 mL DAPI Solution for 20 min at Room temperature or O/N at 4 °C .

SECTION: Staining
17. Rinse in PBSTx and mount in  .