Nov 05, 2023
Wholemount Edu Staining (Zebrafish Larvae)
- Henna Myllymaki1,
- Abigail Elliot1,
- Yi Feng1
- 1Centre for Inflammation Research, Institute for Tissue Repair and Regeneration, Edinburgh BioQuarter, University of Edinburgh
Protocol Citation: Henna Myllymaki, Abigail Elliot, Yi Feng 2023. Wholemount Edu Staining (Zebrafish Larvae). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld9429g5b/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 for larvae between 1-5 dpf. Our analysis is often focused on the skin tissue, but we saw cells in other part of the fish were also stained.
Created: October 22, 2021
Last Modified: November 05, 2023
Protocol Integer ID: 54435
Keywords: zebrafish embryo, evaluating cell proliferation, tissue cultured cell, assays by invitrogen, cultured cell, zebrafish larvae, cell biology, cell proliferation in vivo, wholemount edu staining, cell proliferation, cell, crucial edu injection step, assay, cancer biology, staining protocol, variable edu incorporation efficiency, inconsistent results due to variable edu incorporation efficiency, developmental biology, field of developmental biology, fast staining speed, vivo, standard kit from invitrogen
Funders Acknowledgements:
Wellcome Trust
Grant ID: 100104/Z/12/Z
Cancer Research UK
Grant ID: C38363/A26931
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Abstract
Methods for evaluating cell proliferation are important for research in the field of developmental biology, cancer biology and cell biology. Recent years, Click-iT EdU Assays by Invitrogen is gaining popularity due to its fast staining speed, mild tissue treatment condition, reproducibility and specificity. However, the standard kit from Invitrogen is designed for tissue cultured cells, hence the manufactory provided protocol is optimized for cells cultured on coverslips. EdU staining became a popular method to evaluate cell proliferation in vivo in zebrafish embryos. However, the protocols that we have tested in the past often yield inconsistent results due to variable EdU incorporation efficiency. Here we describe an EdU staining protocol that we optimized for zebrafish larvae, which incorporated a crucial EdU injection step. Our protocol was used successfully by many new students in training, without any issues.
EdU staining
4h 50m
Inject 2nl bolus of 500uM EdU into the yolk of embryos
Note
Reagents for EdU staining can be purchased from - https://www.thermofisher.com/uk/en/home/references/protocols/cell-and-tissue-analysis/protocols/click-it-plus-edu-imaging-protocol.html
embryos were immobilized in 3% methylcellulose in 0.3 x DANEAU's solution (or E3) and returned to induction solution after injection.
30m
Incubate at 28.5°C in the dark for 2.5 hours (this can be between 2 -3 hours, but be consistent between experiments; we have done 2 hours)
2h 30m
Fix in 4% PFA (30min, Room temperature)25 °C
00:30:00
30m
Wash in 0.1% PBT (5min, RT shaking) 30 rpm, 25°C, 00:05:00 repeat 3 times
15m
Wash in 3% BSA, Dilute in PBT (5min, RT shaking) -grams/100ml, i.e. 3g/100ml, 300mg in 10ml.30 rpm, 25°C
5m
Block in 3% BSA, Dilute in PBT (1h, shaking)30 rpm, 25°C, 01:00:00
1h
Incubate in staining solution (as per kit protocol) – we use 250ul per sample (10-15 larvae) 30 min, 30 rpm, 25°C, 00:30:00
250 µL per 10-15 larvae
30m
Immunostaining in dark (EGFP or hRAS)
1d 17h 50m
Wash in PBT (5min, RT shaking x3) 30 rpm, 25°C, 00:05:00 X3
15m
Wash in 5% Goat Serum, Dilute in PBT (5min, RT shaking)30 rpm, 25°C, 00:05:00
5m
Block in 5% Goat Serum, Dilute in PBT (at least 2h, RT shaking)30 rpm, 25°C, 02:00:00
2h
Incubate with anti-GFP Primary Ab 1:200, dilute in 5% Goat Serum in PBT (O/N, +4 °C shaking)
30 rpm, 4°C, 16:00:00 Over night
250 µL 10-30 larvae
Note
GFP (D5.1)XP Rabbit mAB (Cell Signalling Technology 2956)
16h
Wash in PBT (25-30 min, RT shaking) x 8-10 à >4 hours in total!!!30 rpm, 25°C, 00:30:00 X10
5h
Incubate with Secondary Ab [] 1:250, Dilute in 3% Goat Serum in PBT (overnight at +4 °C -- prefered)30 rpm, 4°C, 16:00:00 Or 25C 2 hours
Note
Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen A11008)]
This step over night is preferred, however, 2 hours at RT also works.
16h
Wash in PBT (15 min, RT shaking) x430 rpm, 25°C, 00:15:00 X 4
1h
Stain with Hoechst 33258 Dilute 1 in 1000 in PBT (30mins, RT shaking)30 rpm, 25°C, 00:30:00 in dark
Note
Or other nuclear stain such as DAPI
30m
Wash in PBT (15mins, PBT) x430 rpm, 25°C, 00:15:00 X 4
1h
Embed and Image
10m
Incubate in AF1 solution.
Note
Sample can be stored in the dark for few weeks
10m
Mounting with cover-slip and glass slides.