Virginie Orgogozo

Adult muscle staining protocol

Fly Culture
All were done at 25C. Flies were collected 2-8days after hatching. Flies younger than 2 days old still have larval muscles that cover muscles of Lawrence.

Abdominal Dissection of fresh flies
1. pin the fly in the thorax with ventral face up in PBT in a Sylgard dish.
2. cut the genitalia with iridectomy scissors
3. cut the abdomen along both lateral sides (between ‘white’ and pigmented cuticle) starting from the posterior
4. remove all internal organs with forceps.
5. cut the remaining carcass away.

Abdominal Dissection of stored flies
Flies stored in acetone : gives a lot of fluorescent background with Hoechst. In flies frozen at –80C, the muscles dry out and break. The best technique seems to store flies in EtOH 70% + water + glycerol 5%.
1. rehydrate flies in EtOH 50%, then PBT, 3washes
2. keep flies in PBT for about an hour
3. pin the fly in the thorax with ventral face up in PBT in a Sylgard dish
4. cut the genitalia with iridectomy scissors
5. cut the abdomen along both lateral sides (between ‘white’ and pigmented cuticle) starting from the posterior
6. remove all internal organs with forceps. Use light to make sure you don’t remove the muscles. Try to remove all the internal organs that are on the muscles
7. cut the remaining carcass away.
8. fix in formaldehyde 5% for 10-15min


Preparation for visualisation with polarized light
1. rinses the dissected carcass in EtOH 95% 3x 3min.
2. then EtOH 100% 3x 3min.
3. clear in methylsalicylate 1x 3min (the methylsalicylate and xylene steps have to be done in glass containers because it melts plastic containers).
4. clear in methylsalicylate ON at RT.
5. rinses in zylene 2x 3min.
6. wash in PBT 3x 3min.
7. mount in glycerol 80%.


Preparation for fluorescent microscopy (Hoechst + phalloidin)
1. put the dissected carcass in 5% formaldehyde for 15-20 min.
2. wash 2 times with PBS+Triton X-100  0.5%.
3. incubate 3h at RT or overnight at 4C in phalloidin-Oregon green (1uL, Molecular Probes) +Hoechst (5uL, Molecular Probes) in 400uL PBS-Triton (final concentration=1uM phalloidin, 10ug/mL Hoechst). This solution can be used at least 5 times if stored at 4C.
4. wash once in PBS-Triton 15min.
5. mount in Vectashield mounting medium with muscles on top and carcass on bottom (the reverse gives much less fluorescence in the microscope).

Preparation for fluorescent microscopy (antibodies staining)
6. put the dissected carcass in 5% formaldehyde for 15-20 min.
7. wash 2 times with PBS+Triton X-100  0.5%.
8. wash 1h with PBS+ Triton 0.5% + NGS 5% (TNT-NGS)
9. incubate with primary antibodies ON at 4C (better) or for 2h at RT (a-DNcadherin 1/20, a-FasIII 1/20, a-22C10 1/50 in TNT-NGS)
10. wash 3x20min with TNT-NGS
11. incubate with secondary antibodies 4-5h at RT (mouse-Cy3, 1/250, rabbit-FITC, 1/500 in TNT-NGS) + phalloidin-Oregon green  (1uL, Molecular Probes) +Hoechst (5uL, Molecular Probes) in 400uL TNT-NGS 2-4h at RT. (final concentration=1uM phalloidin, 10ug/mL Hoechst) + TO-PRO-3 1/1000.
12. wash 3 times in PBS-Triton 15min.
13. mount in Vectashield mounting medium with muscles on top and carcass on bottom (the reverse gives much less fluorescence in the microscope).