The goal was to instantly preserve structure and stop biologic degradation through rapid freezing, then more gradually fix the tissue by freeze substitution of M-AA for ice. fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to Indomethacin (Indocid, Indocin) fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, Indomethacin (Indocid, Indocin) as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. Conclusions A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential Indomethacin (Indocid, Indocin) shrinkage, autofluorescence, and tissue immunoreactivity have been problematic. Introduction Sectioned tissue offers a powerful substrate for many cell biologic approaches, such as immunocytochemistry, in situ hybridization, and the recognition of dividing cells. However, these approaches usually require striking a balance between attempts required to preserve structure and the structural artifacts and experimental limitations imparted by those attempts. Covalent changes of epitopes by aldehyde fixatives is definitely a prime example of how attempts to preserve structure effect antigenicity. Further, protocols that work well for an approach such as in situ hybridization do not necessarily translate into ideal conditions for another approach such as immunocytochemistry. A less common but genuine concern is the fixation gradient that occurs when cells are fixed by immersion: cells at the surface will fix more rapidly and more extensively than those in the center of the tissue. We were motivated to address the issue of fixation gradients by our studies of the rodent ocular lens. The lens is a roughly spherical structure that grows throughout the life of the organism by the addition of fresh layers to its surface (lens structure and development examined in [1]). The net result is an onion-like structure, consisting of hundreds to thousands of concentric shells, each consisting of a single generation of differentiated lens dietary fiber cells about 3 microns solid. Like growth rings of a tree, the shells are arranged in a perfect chronological order: oldest at the center, youngest in the periphery. Also like tree growth rings, no layers of lens cells are lost with age. This means that there is a perfect chronological gradient of cells from surface to center, with recently differentiated layers at the surface and cells generated in utero at the center. In an effort to optimize its part as an optical element, the lens is also avascular. Further, only the 1st 50C100 or so layers of lens dietary fiber cells (of the several thousand present in the human lens) maintain organelles. This means that the majority of dietary fiber cells in the adult lens spend a lifetime without any of the functions imparted by organelles, including mitosis, translation, and transcription, making them a superb model for cellular ageing. Finally, the lens must elevate its index CEACAM8 of refraction well above that of the surrounding aqueous media, and does so by elevating cytoplasmic protein concentrations to a level higher than those of some other cell type. These.