Comparison of fixatives for maximal retention of radiolabeled glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated [35S]-sulfate.

Abstract

Previous studies have used [35S]-sulfate as a specific marker to autoradiographically localize sulfated glycosaminoglycans, proteoglycans, and glycoproteins. Embryonic chicks were labeled with [35S]-sulfate, followed by previously reported routine fixation and processing techniques. Subsequent processing revealed loss of radiolabeled macromolecules and retention of unincorporated label in the tissue, using these procedures. Biochemical analysis after various fixation and processing procedures demonstrated that an additional agent, such as cetylpyridinium chloride, was necessary in the fixative to retain the highly aqueous soluble sulfated macromolecular components. Molecular sieve chromatography was used to monitor digestate solutions for the identity of glycosaminoglycans and proteoglycans as indicated by selective enzymatic removal. Retained unincorporated [35S]-sulfate could be completely removed by rinsing the tissue in dehydration solutions containing exogenous sodium sulfate. This new procedure ensures the quantitative retention of sulfate labeled macromolecules in fixed tissue with the complete removal of unincorporated radiotracer, both of which are necessary for meaningful autoradiography.