Key steps in the separation of the membrane from the overlying soft tissues. (a) Mouse with dorsal air pouch just before dissection. (b) A small incision is made into the dorsal skin overlying the pouch. This incision is just deep enough to expose the apex of the pouch membrane. (c) The membrane is punctured with a scalpel or needle. (d) The pouch content is lavaged with 2 ml phosphate-buffered saline. If the opening is enlarged sufficiently, the lavage can be performed under direct visualization. (e-i) Using blunt dissection with curved clamps or curved scissors, the pouch membrane is separated meticulously from the overlying skin. The instrument follows a path of least resistance between the membrane and the overlying soft tissues. (j) Parts of the membrane adhering to the more caudal skin can be clipped off with fine scissors. Finally, the membrane collapses on the dorsum of the animal. (k, l) The membrane is then grasped with forceps, elevated and cut at the base with scissors. (m) The gelatinous appearing dissected membrane (arrow) adhering to the forceps just before homogenization in Trizol medium.