Quick-freezing and freeze-drying in preparation for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cell.
Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.
D000818:Animals; D005260:Female; D005612:Freeze Drying; D005615:Freezing; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D007328:Insulin / Q000032:analysis; D007515:Islets of Langerhans / Q000032:analysis / Q000648:ultrastructure*; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D013048:Specimen Handling / Q000379:methods*
D007328:Insulin