Quantification of tracheal density in IFMs

Histology of tracheal branches in adult IFMs

1. Anesthetize flies and remove the head, abdomen, wings and legs from the thorax using sharp 
   scissors or forceps.
2. Collect the thoraxes in a well with PBT (PBS + 0.3% Triton X-100) on ice.
3. Remove PBT.
4. Fix with 500 μL 4% PFA in PBT for 20 min.
5. Remove fixation solution.
6. Rinse the thoraxes once with PBT.
7. To bisect the thorax, place it on double-sided sticky tape and cut transverse using a sharp microtome blade. Ideally the thorax is cut in one movement and the blade is not moved back and forth after the cuticle has been cut.
8. Transfer thorax halves to blocking solution (5% BSA in PBT) and incubate at RT for 1 hour or overnight at 4 °C.
9. Dilute Phalloidin-TRITC 1:300 in PBT and incubate for 1 hour at RT.
10. Wash 3x 10 min with PBT. Prepare in the meantime glass slides with spacers.
11. Mount in Mowiol (Sigma).
12. Seal with nail polish.

Sauerwald J, Backer W, Matzat T, Schnorrer F & Luschnig S (2019) Matrix metalloproteinase 1 
modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles. eLife 8:

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