Mar 26, 2026
Immunostaining of Drosophila embryos
- Artem Ilin1
- 1SU
Protocol Citation: Artem Ilin 2026. Immunostaining of Drosophila embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11e7yvr2/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: December 11, 2025
Last Modified: March 26, 2026
Protocol Integer ID: 234745
Keywords: drosophila embryo
Abstract
A protocol for Drosophila embryos immunostaining
Materials
Sodium hypochlorite 6-14% (has to be fresh, at least from this year)
Baskets for embryo collection from juice plates
PBS
PBST: PBS + 0.3% Triton-X 100. For 50 ml: 48.5 ml PBS + 1.5 ml 10% Triton-X.
PBSBT: PBST + 0.5% (by weight) BSA. For 50 ml: dissolve 0.25 g of BSA in ~10-20 ml of PBST (place on rotator). After BSA is dissolved, adjust volume with PBST up to 50 ml. Filter using Filtropur S 0.2 filter and a 50 ml syringe.
Methanol
Heptane
VectaShield
Glass slides and covers
Troubleshooting
Dechorionation and fixing
52m
Transfer aged embryos from juice plate(s) to the basket, wash with water and put into the container with 5x diluted sodium hypochlorite 6-14% for 2 minutes. Check dechorionation under the binocular microscope.
15m
Wash the basket with water, transfer the embryos from the net to the glass vial with containing 1x PBS using the brush.
5m
Wait until embryos go down, remove the PBS, add 3.6 mL of 1x PBS. Add 4 mL of heptane and 400 µL of 37% formaldehyde.
3m
Place on shaker 350 rpm, 00:25:00
25m
Remove the lower phase, add 5 mL of methanol. Shake vigorously by hand for a bit more than 1 minute.
2m
Wait until the majority of embryos sink. Remove the liquid.
2m
Add 5 mL of methanol, shake briefly, wait until embryos sink. Transfer the embryos in methanol to 1.5 ml eppendorf tube.
Immunostaining: primary antibodies
1h 25m
Transfer the embryos to eppendorf. Add 500 µL of methanol and 500 µL of H2O MQ. Place on rotator for 00:10:00
10m
Transfer to 1.5 ml eppendorf tube, wash 3x 00:05:00 with 1 mL PBST.
15m
Wash 4x 00:15:00 with 1 ml PBSBT
1h
Dilute primary antibodies (according to antibody reference) in 500 µL of PBSBT for each sample. Add antibody mix to your samples. Place at rotator in the cold room overnight.
Immunostaining: secondary antibodies
3h
Remove the primary antibodies from your samples and save them for a potential reuse.
Wash embryos 3x00:05:00 with PBSBT. Wash 3x 00:15:00 with PBSBT.
1h
Add secondary antibodies (fluorescent) according to primary (organism) diluted 1:800 in 500 µL PBSBT. Cover the tubes in foil and place on rotator for 02:00:00 Room temperature
2h
Wash 3x00:05:00 and 4x00:20:00 with PBSBT. Wash 00:30:00 with PBS
Add 50 µL of VectaShield mounting medium (with DAPI). Mount on glass slides.