Preparation of the Indirect Flight Muscle (IFM) of Drosophila melanogaster for high pressure freezing

Freezing tissue for high pressure freezing must be fast. Excision of the tissue might be done by needle biopsy, which is favourable for tissue of mammals (e.g. muscle). Dissection of small beings as Drosophila dissection must be fast and can be done the best by dissection in buffer. Mangling of tissue is likely. So in the case of the IFM (the muscle just beneath the thorax) can be kept intact due to the covering sheath of chitin. The specimen platelet (diameter 1.2 mm, depth 200 micrometres) cavity is filled with a good-to-vitrify liquid (non-osmotic: 1-hexadecene, dextran; osmotic: sucrose, buffer) and immediately frozen at 2100 bar in liquid nitrogen (-196°C) using a Leica EmPact High Pressure Freezer.

Freeze substitution