The Good, the Bad and the Ugly: Task-Specific Fixation for Connective Tissues

Abstract

Tissues with high water content, mediated by highly concentrated proteoglycans, are exceptionally resistant to ultrastructural preservation. Due to their net negative charge, proteoglycans allow the retention of substantial water and also interact strongly with cells, collagens and other molecules to form a hydrated, integrated, and resilient matrix. Proteoglycan content varies within specific tissue compartments, but is ubiquitous to all connective tissue matrices. A significant challenge presented to the Microscopist is in the stabilization of this fragile and extensive proteoglycan network, whose structure is dependent on retained water which itself must be removed to be compatible with most embedding media. Furthermore, and regrettably, proteoglycans are largely (70%) extracted during conventional glutaraldehyde/Osmium fixation. The remaining structures (cells and collagen fibrils) are separated by large open spaces previously occupied by the extensive proteoglycan matrix and water. Cryostabilization (high pressure freezing followed by freeze-substitution) is unique in its ability to stabilize the intimate proteoglycan/collagen/cell associations; however particularly in skin, many landmark structural entities are masked by the retained proteoglycans and cannot be visualized. HPF/FS is triumphant in revealing the ultrastructure of smaller organisms; however the successes in cooling exceedingly hydrated tissue ice-crystal free are so rare that biopsies must not be sacrificed to this procedure. Certainly, the few images of well cryo-preserved connective tissue need consideration when deliberating spatial relationships in this difficult tissue (1).