Ab-pupal-muscles

Virginie Orgogozo

Pupae muscle staining protocol

Staging pupae
Select white pupae (=puparium formation) and let them grow at 25C for x hours = x h APF (after puparium formation).

Carcass dissection
1. put the pupae on its lateral side on double-sided sticky tape on a slide.
2. cut the pupae in half using a double side razor blade. Each side of the blade is generally sharp enough to cut 10-15 pupae.
3. grab the dorsal epidermis by the thorax with a forceps and transfer it without the pupal case to a well filled with PBT. Transfer also the ventral epidermis to another well. Look for the gonad to distinguish male from female pupae (both are attached and pear-shaped for the male, they are smaller, round-shaped with the ovarioles already formed for the female).
4. hold the dissected tissue by the thorax with a forceps and remove the pair of tracheal branches located in the abdomen with another forceps.
5. hold the dissected tissue by the thorax with a forceps and gently blow PBT into the cavity to remove the tissues (with a P20 pipette set on 5uL). Do it very carefully so that the muscles stay attached to the epidermis.
6. transfer the epidermis to a well for staining.

Fluorescent antibodies staining
1. put the dissected carcass in 4% formaldehyde for 10-15 min.
2. wash 2 times with PBS+Triton X-100 0.5%.
3. wash 1 hour with PBS+ Triton 0.5% + NGS 5% (TNT-NGS)
4. incubate with primary antibodies ON at 4C (22C10 1/50, B3-tubulin 1/1500 in TNT-NGS)
5. wash quickly with TNT-NGS
6. wash 2x30min with TNT-NGS
7. incubate with secondary antibodies 4-5h at RT (mouse-Cy3, 1/250, rabbit-FITC, 1/500 in TNT-NGS)
8. wash quickly with TNT-NGS
9. wash 45min with TNT-NGS
10. wash 20min with TNT-NGS+Hoechst (5uL in 400uL TNT-NGS (final concentration=10ug/mL).
11. on a slide, glue one coverslip as a left spacer and one as a right spacer with nail polish. Mount in Vectashield mounting medium with muscles on top and carcass on bottom (the reverse gives less fluorescence in the microscope).