Chemical and physical fixation of the lung. Alveolar epithelial type II cells were studied by cTEM either after chemical (A and B) or after physical (C and D) fixation. The overview in A shows a well-preserved type II cell from a newborn rat lung fixed by instillation of 1.5% GA, 1.5% PFA in Hepes buffer and processed according to Table 1. Lamellar bodies (LB), nucleus (Nu) and mitochondria (Mt) are well preserved. At a higher magnification, details of the endoplasmic reticulum (ER) as well as an ER related multivesicular transport vesicle (MvTV) can be visualized. The overview in C shows a well-preserved type II cell from an adult rat lung. A small piece of tissue was cut from the whole lung, put in a syringe with 1-hexadecene and air was extracted from the tissue block by negative pressure. Afterwards, the specimen was high-pressure frozen (Leica EMPact 2.0, Leica, Vienna, Austria), freeze-substituted with acetone containing 1% osmium tetroxide (AFS 2.0, Leica, Vienna, Austria) and embedded in epoxy resin. Most likely due to the lack of uranyl acetate during freeze-substitution the lamellar bodies are not well preserved, with almost complete loss of the surfactant material, only the limiting membrane can be seen. However, the ultrastructure of other organelles like multivesicular bodies (MvB) is highly increased (D) due to the excellent preservation of the membrane structures (Me). Since this is the first description of high-pressure frozen lung tissue, systematic studies are needed to determine the ideal processing both for conventional and immuno TEM. Bars = 1 μm (A, C), 250 nm (B, D).