Feb 24, 2026

Public workspaceDrosophila Fixation for Pico-C

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz9ro5gx1/v1
  • Noura Maziak1,2,
  • Juanma Vaquerizas1,2
  • 1MRC Laboratory of Medical Sciences, London, W12 0HS, UK;
  • 2Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0HS, UK
Noura Maziak
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz9ro5gx1/v1
Protocol Citation: Noura Maziak, Juanma Vaquerizas 2026. Drosophila Fixation for Pico-C. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9ro5gx1/v1
Manuscript citation:
Maziak N, Zhang Y, Groll F, Brown HE, Madich A, Kaur Y, Harrison MM, Zhou J, Vaquerizas JM. 3D Genome Reorganization Foreshadows Zygotic Genome Activation in Drosophila. Nature Genetics. In press.
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: November 08, 2025
Last Modified: February 24, 2026
Protocol Integer ID: 231850
Keywords: drosophila fixation for pico, drosophila fixation, drosophila, pico
Funders Acknowledgements:
Medical Research Council, UK
Grant ID: MC_UP_1605/10
Academy of Medical Sciences and the Department of Business, Energy and Industrial Strategy
Grant ID: APR3\1017
Abstract
Fly embryo fixation for Micro-C/Pico-C.
Materials
General:
  • 13% sodium hypochlorite (bleach)
  • 2M Tris-HCl pH 7.5
  • PBS
  • Triton X-100
  • 16% formaldehyde (fresh from 1ml ampules) (Pierce 28906)
  • EGS (ethylene glycolbis(succinimidyl succinate)) (Thermo Scientific 21565)

Optional:
  • Corning cell strainer CLS431752; pore size 100 μm, sterile, pkg of (individually wrapped)
  • Dissecting needle with plastic/wood handle (example) [This is a personal preference. I find that this gives me much more control when sorting through embryos. Because we do this under the fluorescent microscope to capture exact nuclear cycles, this provide a bit more flexibility than a paint brush]
Troubleshooting
Drosophila Fixation for Pico-C
Prepare PBST (PBS-0.5% Triton). For 40 mL volume, add 2 mL of 10% Triton X-100 stock solution to 38 mL of PBS. The addition of Triton helps minimize embryo loss during washes and can also be included in the Tris buffer during quenching if needed.
Collect embryos on agar plates with acetic acid.
Rinse embryos with water into a Nitex nylon mesh.
Note: We commonly now use cell strainers with large pore sizes.
Dechorionate with bleach solution (half bleach half sterile water for a total of 15 mL) for 3 min. Embryos will float when dechorionated. If they do not, let them sit for a few more seconds until they do.
During this time take out EGS and store in the dark at room temperature until you need it later.
Note: EGS is moisture-sensitive. Equilibriation to room temperature before opening helps avoid moisture condensation onto the product.
Rinse embryos thoroughly with slowly running tap water, including the sides of the Nitex mesh basket.
Sit the Nitex mesh basket in a small petri dish filled with PBST. The embryos will largely remain on the surface. Use a 1 ml pipette to collect embryos and transfer to a 15 ml Falcon tube. Once all embryos are transferred, remove any excess PBST, leaving only 2 mL. To this, add 6 mL heptane and 600 μl 16% formaldehyde (from a freshly opened ampule). Close the tube tightly and start a timer.
Shake the tube or vortex on high for 30s. The solution should turn milky white.
Agitate the vial (e.g. 30rpm on a tube rotator) for 15 min.
At precisely 15 min after the start of fixation, add 3.7 mL of 2M Tris-HCl pH 7.5 to quench, shake to mix or vortex for 30s, and rotate for 5 min.
Centrifuge tubes at 500xg for 1 minute to separate the liquid fractions and collect the embryos at the bottom of the tube. The upper organic phase will have the consistency of shampoo. This is okay.
Remove as much of the upper organic phase as possible. We find this easiest using a disposable plastic transfer pipette / dropper, but you can also pipette it out. Remove as much of the lower aqueous phase as possible without losing any embryos (embryos will stick to plastic at this point- avoid contact).
Wash embryos multiple times with ice-cold PBST. During the first few washes, use gentle pressure when pipetting to help dislodge embryos that may stick to the tube walls. Once the embryos begin to float freely, they can be carefully transferred to a 1.5 mL Eppendorf tube.
Transfer embryos in PBST to an 1.5 mL Eppendorf tube on ice. Wash 2X more in 1 ml ice-cold PBS-Triton.
Store embryos at 4°C until all batches are ready, then combine all batches.
Freshly prepare the second crosslinkers. Mix thoroughly 13.7 mg (0.0137 g) EGS in 100ul DMSO for a 300mM stock
CrosslinkerMWSpacerStockWorking Solution
EGS 456.36 16.1 300 mM in DMSO 3 mM in PBST
Note: Before adding the crosslinker – bring samples to room temperature to prevent DMSO from solidifying (freezing point 19 °C); second, EGS can form a white cloudy precipitate when diluted in small volumes. This should not affect the reaction – but can possibly be avoided by making a more dilute stock in DMSO (like using 200μL, would mean you add 20μL in next step). Information can also be found here.

Crosslink embryos in 10 mL PBST by adding 10 µL of the 300 mM EGS stock to obtain a final concentration of 3 mM, and incubate for 45 min at room temperature with gentle rotation.
Quench by adding 3.7 mL of 2M Tris pH 7.5 for 5 min, rotating.
Wash 2X with PBST and transfer to 1.5ml tube. Store embryos on ice.
Sort embryos for desired stage.
After sorting, embryos were snap-frozen in liquid nitrogen or dry ice after removing as much supernatant as possible and stored at -80 C.
Protocol references
Adapted from:

Blythe, S. A., & Wieschaus, E. F. (2016). Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis. eLife, 5, e20148. https://doi.org/10.7554/eLife.20148

Hsieh, TH., Fudenberg, G., Goloborodko, A. et al. Micro-C XL: assaying chromosome conformation from the nucleosome to the entire genome. Nat Methods 13, 1009–1011 (2016). https://doi.org/10.1038/nmeth.4025

Ing-Simmons, E., Vaid, R., Bing, X. Y., et al. (2021). Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nature Genetics, 53, 487–499. https://doi.org/10.1038/s41588-021-00799-x
Acknowledgements
We thank Elizabeth Ing-Simmons for help establishing the fixation protocol and for her invaluable guidance.