%0 Journal Article
%T Chemical Separation of Fixed Tissue Using Thermolysin
%A Anahita Dua
%A Sapan S. Desai
%J Journal of Histology
%D 2013
%R 10.1155/2013/643670
%X Thermolysin is a metallopeptidase used to cleave peptide bonds at specific junctions. It has previously been used to cleave specific amino acid sequences found at the junction of the sensory epithelium and underlying stroma of unfixed otolithic organs of the vestibular system. We have used thermolysin to separate sensory epithelium from the underlying stroma in fixed cristae ampullares of mouse, rat, gerbil, guinea pig, chinchilla, and tree squirrel, thus removing the saddle-shaped curvature of the sensory organ and creating a flattened sensory epithelium preparation. This permits visualization of the entire sensory organ in a single mount and facilitates proper morphometric analysis. 1. Introduction Thermolysin, otherwise known as protease type X, is found in Bacillus thermoproteolyticus [1]. Thermolysin is a metallopeptidase and cleaves peptide bonds at the N-terminal of hydrophobic amino acids including alanine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine [2]. Thermolysin was first characterized by Matsubara et al. [3] and subsequently used in unfixed otolith organ tissue to cleave specific amino acid sequences found at the junction of the sensory epithelium and underlying stroma [3, 4]. Suzuki et al. [4] were able to use thermolysin to cleave proteins anchoring the otolithic membrane to the tectorial membrane [4]. After centrifugation, they were able to separate the gelatinous layer from the otolithic stones. Saffer et al. [5] used thermolysin to separate the sensory epithelium from nonsensory epithelium in unfixed utricular maculae of rats [5]. Thermolysin has also been used to separate unfixed human keratinocytes, cultured intestinal epithelial cells, cultured cochlear spiral ganglion neurons, and pancreatic islet cells [6¨C10]. Thermolysin has not previously been used to separate fixed tissue, and there is concern that the use of fixatives such as glutaraldehyde, formaldehyde, and acrolein may change the protein structure such that it is no longer amenable to cleavage by thermolysin. Despite this concern, we have used thermolysin to separate sensory epithelium from the underlying stroma in fixed cristae ampullares of mouse, rat, gerbil, guinea pig, chinchilla, and tree squirrel (Figure 1). This resulted in removal of the extracellular matrix that imparts the three-dimensional saddle shape of the sensory organ and the creation of a flattened sensory epithelium preparation. This two-dimensional view was desired to improve visualization of the entire sensory epithelium at once and to allow more accurate
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