Aug 29, 2024
Immunofluorescent staining of dopaminergic neurons in brains of D. melanogaster
- Natalie Kaempf1,2,3,
- Uli Pech1,2,
- Patrik Verstreken1,2
- 1VIB-KU Leuven Center for Brain & Disease Research, 3000 Leuven, Belgium;
- 2KU Leuven, Department of Neurosciences, Leuven Brain Institute, 3000 Leuven, Belgium;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Natalie Kaempf, Uli Pech, Patrik Verstreken 2024. Immunofluorescent staining of dopaminergic neurons in brains of D. melanogaster. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyweyevx9/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 17, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103595
Keywords: ASAPCRN, Immunohistochemistry, dopaminergic neurons, Drosophila brains
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000430
EMBO long-term postdoctoral fellowship
Grant ID: ALTF_299-2019
Research project, FWO Vlaanderen
Grant ID: G0A5219N
Research project, FWO Vlaanderen
Grant ID: G0B8119N
Methusalem project
Grant ID: METH/21/05 (3M210778)
Research project, KU Leuven Parkinson Fonds
Grant ID: EQZ-PARFON-O2010
Opening the Future grant, Leuvens Universiteitsfonds (LUF)
Grant ID: EQZ-OPTFUP-O2010
Research project, FWO Vlaanderen
Grant ID: G031324N
Abstract
This protocol is used to visualize for dopaminergic neurons in Drosophila brains, but can be adapted for other targets as well.
Materials
Rabbit polyclonal anti-TH, Millipore, Cat# AB152, RRID: AB_390204
Mouse monoclonal anti-DLG, DSHB, DSHB Cat# 4F3 anti-discs large; RRID: AB_528203
Goat anti-Rabbit IgG Alexa Fluor‱ 488, Life Technologies, Cat# A-11034 RRID: AB_2576217
Goat anti-Mouse IgG Alexa Fluor‱ 555, Life Technologies, Cat# A-21424 RRID: AB_141780
RapiClear 1.47, Sunjin Lab, Cat# RC147001
Triton X-100 Solution, Sigma Aldrich, Cat# 93443-100ML
Formaldehyde solution (37 wt. % in H2O) F1635-500ml, Sigma-Aldrich
Dissection of fly brains, fixation, block and primary antibody incubation
Dissection of fly brains, fixation, block and primary antibody incubation
1h 30m
1h 30m
Prepare fresh 3.7% paraformaldehyde in 1x PBS, 0.2% Triton X-100 (PBX), 500 µL per genotype and store On ice .
Dissect fly brains in ice-cold PBS under stereomicroscope, collect with a PBX coated pipette and transfer in the 3.7% paraformaldehyde solution.
Store the dissected fly brains in paraformaldehyde solution on ice, but do not keep fly brains longer than 01:00:00 On ice
1h
Incubate the dissected fly brains in paraformaldehyde solution on rotation wheel for 00:30:00 at Room temperature
30m
Take the paraformaldehyde solution off and wash a first time with PBX 0.2% (500 µL ) and take it
off right away
Wash 3 times with PBX 0.2% (500 µL ) for 15 min on a rotator at Room temperature
Block with 10% NGS in PBX (500 µL ) for 01:00:00 on rotator at Room temperature
1h
Add primary antibody (in 500 µL of 10% NGS in PBX) put it on rotation wheel at 4 °C 48:00:00
2d
Use rabbit a-TH, Sigma, 1:200, and mouse a-DLG, DSHB, 1:100
secondary antibody incubation
secondary antibody incubation
2d
2d
Take the primary antibody solution off and wash a first time with PBX 0.2% (500 µL ) and take
it off right away
Wash 3 times with PBX 0.2% (500 µL ) for 15 min on a rotator at Room temperature
Add secondary antibody (in 500 µL of 10% NGS in PBX), cover with tinfoil and put it on rotation wheel at 4 °C Overnight
2d
Use Goat anti-Rabbit IgG Alexa Fluor‱ 488, Life Technologies, and Goat anti-Mouse IgG Alexa Fluor‱ 555, Life Technologies, at 1:500 dilution.
Mounting
Mounting
15m
15m
Take the secondary antibody solution off and wash a first time with PBX 0.2% (500 µL ) and take
it off right away
Wash 3 times with PBX 0.2% (500 µL ) for 15 min on a rotator at Room temperature
prepare slides:
clean slides with EtOH
place 2 book binder rings on top of each other
fill book binder rings with 0.075% PLL solution, leave minimum 00:15:00 , take the solution off and leave a thin layer to dry
15m
Mount fly brains by pipetting the brains next to the book binders, fill the book binder chamber with PBX and transfer the brains individually with forceps to the book binder chamber, orient the brains correctly with anterior facing up (antenna lobe up) and softly push down until they stick
remove PBX
add 8 μl of mounting medium (RapiClear 1.47) by gently pipetting on top of brains
add glass coverslip, seal with nail polish and let cure overnight at 4 °C in dark