Dec 11, 2023

Public workspaceEdU Staining Protocol in juvenile F. hepatica

PLOS Pathogens
DOI
dx.doi.org/10.17504/protocols.io.eq2lyjnrrlx9/v1
  • 1Queen's University Belfast
Paul McCusker
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DOI: dx.doi.org/10.17504/protocols.io.eq2lyjnrrlx9/v1
Protocol Citation: Paul McCusker, Duncan Wells, Rebecca Armstrong, Emily Robb, Paul McVeigh, Nathan Clarke, Erica Gardiner, Erin McCammick, Aaron Maule 2023. EdU Staining Protocol in juvenile F. hepatica. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjnrrlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 31, 2023
Last Modified: December 11, 2023
Protocol Integer ID: 85723
Keywords: Fasciola, EdU, Proliferative cells
Funders Acknowledgements:
LIVER FLUKE MOTOR FUNCTION AND PARASITE CONTROL: EXPLOITING A 'TARGET VALIDATION TOOLBOX' AS A DRUG SCREEN-INTERFACE FOR FLUKICIDE DISCOVERY
Grant ID: BB/K009583/1
Probing in vivo parasite biology in vitro
Grant ID: NC/N001486/1
Exploiting stem cell biology for liver fluke control
Grant ID: BB/T002727/1
Abstract
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.
Materials
CS50 - 50% ReagentChicken Serum, New Zealand OriginThermo FisherCatalog #16110082 and 50% ReagentRPMI 1640 Medium, no phenol redThermo FisherCatalog #11835105 with x1 ReagentAntibiotic Antimycotic Solution (100×), StabilizedMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5955

PBSTx - 1X PBS + 0.3% ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
4%FA - Amount500 µL ReagentFormaldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #F8775-25ML with Amount4 mL PBSTx

4%PFA - Add Amount8 g Paraformaldehyde to Amount100 mL millipore water, stir/heat in a flow hood for 1 h between Temperature55 °C and Temperature60 °C on hotplate (NB. must NOT go above 60oC; if it does, start again). Following 1 h heating turn off heat and add NaOH dropwise until solution goes clear before adding Amount100 mL 2X PBS. and mix. Store at Temperature4 °C for 2 weeks orTemperature-20 °C in aliquots for 6 months.
EdU Detection Solution

ABCDE
Reagent 200 (µl)1 ml (µl)5ml (ml)10ml (ml)
PBS1587893.9457.89
100mM Copper Protectant in Water (Store 4oC)2100.050.1
10mM AlexaFluor (488 or 545)/6-FAM azide0.210.0050.01
500mM Ascorbic Acid (Make fresh – 88 mg in 1 ml PBS)4020012
Copper protectant - ReagentCopper(II) sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #451657
Alexafluor 488 - ReagentAzide-fluor 488Merck MilliporeSigma (Sigma-Aldrich)Catalog #760765
6-FAM azide - Reagent6-Fam AzideMetabion
DAPI solution - 1:1000 of Concentration1 mg/mL stock in PBSTx
Protocol materials
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
ReagentRPMI 1640 Medium, no phenol redThermo FisherCatalog #11835105
ReagentAzide-fluor 488Merck MilliporeSigma (Sigma-Aldrich)Catalog #760765
ReagentCopper(II) sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #451657
ReagentChicken Serum, New Zealand OriginThermo FisherCatalog #16110082
Reagent6-Fam AzideMetabion
ReagentAntibiotic Antimycotic Solution (100×), StabilizedMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5955
ReagentFormaldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #F8775-25ML
ReagentVECTASHIELD Mounting MediumVector LaboratoriesCatalog #H-1000
ReagentVECTASHIELD Mounting MediumVector LaboratoriesCatalog #H-1000
Safety warnings
EdU Mutagen
As a thymidine analogue EdU is known for germ cell mutagenicity and reproductive toxicity. Particular care should be taken when disposing of the chemical and around pregnant mothers.
In vitro addition of EdU to juvenile F. hepatica
In vitro addition of EdU to juvenile F. hepatica
1d
1d
Juvenile liver fluke are cultured in 50% Chicken serum (CS50) as described in McCusker et al. (2016).
Amount10 µL of Concentration10 millimolar (mM) 5-Ethynyl-2'-deoxyuridine (EdU) is mixed with Amount190 µL pre-warmed CS50 and added to worms for between 6 and 24 h.
Step case

Stain immediately following EdU incubation
15 steps

Fixation
Fixation
Add Amount20 µL 4% FA/4% PFA to a petri dish.
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.
Add 4% FA/4% PFA to float coverslip. Lift coverslip and move worms into 1.5 mL eppendorf with Amount1 mL 4% FA/4% PFA. Place tube on a rotator for further 10 min (4%FA)/2 h (4%PFA) at TemperatureRoom temperature OR O/N atTemperature4 °C .

OPTIONAL Storage Step
OPTIONAL Storage Step
Wash in Amount1 mL PBSTx for 10 min.

Wash in Amount1 mL 1:1 PBSTx:Methanol for 10 min.

Store in Amount1 mL methanol at Temperature-20 °C until required.

Rehydrate in Amount1 mL 50:50 PBSTx:Methanol for 10 min.

Staining
Staining
x2 washes in Amount1 mL PBSTx for 10 min each.

Incubate in Amount1 mL fresh Proteinase K Solution for 15 min at TemperatureRoom temperature .

Postfix in Amount1 mL 4%FA for 10 min.

Wash in Amount1 mL PBSTx for 10 min.
Incubate in EdU Detection Solution for 30 min at TemperatureRoom temperature . NB: cover with tinfoil from this point on.

x2 washes in Amount1 mL PBSTx for 5 min each
Incubate in Amount1 mL DAPI Solution for 20 min at TemperatureRoom temperature or O/N at Temperature4 °C .

Rinse in PBSTx and mount in ReagentVECTASHIELD Mounting MediumMetabionCatalog #H-1000 .

Protocol references
McCusker, P., McVeigh, P., Rathinasamy, V., Toet, H., McCammick, E., O'Connor, A., Marks, N. J., Mousley, A., Brennan, G. P., Halton, D. W., Spithill, T. W., & Maule, A. G. (2016). Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro. PLoS Neglected Tropical Diseases, 10(9), [e0004994]. https://doi.org/10.1371/journal.pntd.0004994