Journal
JOURNAL OF MICROSCOPY-OXFORD
Volume 212, Issue -, Pages 13-20Publisher
BLACKWELL PUBLISHING LTD
DOI: 10.1046/j.1365-2818.2003.01225.x
Keywords
electron microscopy; freeze substitution; gastric parietal cell; HeLa cell; high-pressure freezing; immunogold labelling; primary culture
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Funding
- NIDDK NIH HHS [DK10141, DK38972] Funding Source: Medline
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We describe a procedure for high-pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate. Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly-L-lysine-coated planchettes. This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells. Furthermore, the yield of well-frozen cells approaches 100% for every cell type we have tried so far. In this report, we show well-preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands. Immunogold labelling of H+/K+-ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin. The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24-28 h on the planchettes retain their responsiveness to stimulation with histamine.
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