TITLE: Immunofluorescent staining of dopaminergic neurons in brains of D. melanogaster
AUTHORS: Natalie Kaempf, Uli Pech, Patrik Verstreken

[DESCRIPTION]
This protocol is used to visualize for dopaminergic neurons in Drosophila brains, but can be adapted for other targets as well.

[STEPS]
SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
1. Prepare fresh 3.7% paraformaldehyde in 1x PBS, 0.2% Triton X-100 (PBX), 500 µL per genotype and store on ice.  

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
2. Dissect fly brains in ice-cold PBS under stereomicroscope, collect with a PBX coated pipette and transfer in the 3.7% paraformaldehyde solution. 
SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
2.1. Store the dissected fly brains in paraformaldehyde solution on ice, but do not keep fly brains longer than 60 min on ice

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
3. Incubate the dissected fly brains in paraformaldehyde solution on rotation wheel for 30 min at Room temperature

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
4. Take the paraformaldehyde solution off and wash a first time with PBX 0.2% (500 µL) and take it
off right away

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
5. Wash 3 times with PBX 0.2% (500 µL) for 15 min on a rotator at Room temperature

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
6. Block with 10% NGS in PBX (500 µL) for 60 min on rotator at Room temperature

SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
7. Add primary antibody (in 500 µL of 10% NGS in PBX) put it on rotation wheel at 4 °C 2880 min

SECTION: secondary antibody incubation
8. Take the primary antibody solution off and wash a first time with PBX 0.2% (500 µL) and take
it off right away

SECTION: secondary antibody incubation
9. Wash 3 times with PBX 0.2% (500 µL) for 15 min on a rotator at Room temperature
SECTION: secondary antibody incubation
10. Add secondary antibody (in 500 µL of 10% NGS in PBX), cover with tinfoil and put it on rotation wheel at 4 °C 2880 min
SECTION: Mounting
11. Take the secondary antibody solution off and wash a first time with PBX 0.2% (500 µL) and take
it off right away
SECTION: Mounting
12. Wash 3 times with PBX 0.2% (500 µL) for 15 min on a rotator at Room temperature
SECTION: Mounting
13. prepare slides: 
SECTION: Mounting
13.1. clean slides with EtOH
SECTION: Mounting
13.2. place 2 book binder rings on top of each other
SECTION: Mounting
13.3. fill book binder rings with 0.075% PLL solution, leave minimum 15 min, take the solution off and leave a thin layer to dry

SECTION: Mounting
14. 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
SECTION: Mounting
15. remove PBX
SECTION: Mounting
16. add 8 μl of mounting medium (RapiClear 1.47) by gently pipetting on top of brains  
SECTION: Mounting
17. add glass coverslip, seal with nail polish and let cure overnight at 4 °C in dark
SECTION: Dissection of fly brains, fixation, block and primary antibody incubation
7.1. Use rabbit a-TH, Sigma, 1:200, and mouse a-DLG, DSHB, 1:100
SECTION: secondary antibody incubation
10.1. Use Goat anti-Rabbit IgG Alexa Fluor‱ 488, Life Technologies, and Goat anti-Mouse IgG Alexa Fluor‱ 555, Life Technologies, at 1:500 dilution.