A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling.
Ultracryotomy of fixed specimens in combination with immunogold labeling is widely used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible. Normally, sections are cut and labeled within 1 day to prevent possible loss or redistribution of soluble antigens within the sections. An increasing demand for more sections and multiple labeling protocols prompted us to investigate the extent to which ultrathin cryosections can be stored. This would render the time spent behind an ultracryomicrotome more efficient and would allow immunogold labeling at a later stage. We investigated whether gelatin plates, 2.3 M sucrose, or 1.0% methyl cellulose/1.2 M sucrose can be used to store thawed frozen sections for a longer period of time. Ultrathin sections of mildly fixed tissue and cultured cells were stored for up to 6 months before immunogold labeling. The preservation of the ultrastructure of stored sections was excellent and was similar to that of immediately processed sections. Importantly, prolonged storage did not affect the labeling intensity.
Griffith
Janice M
JM
Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, Utrecht, The Netherlands.
Posthuma
George
G
D000418:Albumins / Q000378:metabolism; D000681:Amylases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005629:Frozen Sections; D007150:Immunohistochemistry / Q000379:methods*; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility
D000418:Albumins; D000681:Amylases