Improving structural integrity of cryosections for immunogold labeling.

摘要: Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution this approach. We found step during which sections thawed and transferred from cryochamber supporting film on an EM grid is most critical preservation. Surface tension transfer medium, spread thawing, can easily damage their structure by overstretching. By substituting mixture methylcellulose sucrose conventional we were able alleviate problem overstretching, thus improving greatly integrity thin cryosections. Also, extraction after thawing causes damage, particularly when cross-linking deficient. Incorporation uranyl acetate in medium then further help maintain immunolabeling procedure. Excellent ultrastructure was featured picked up dried directly methylcellulose/uranyl mixtures. Such preparations provide new insight into subcellular details efficient back-up immunolabeled respect morphology. fresh frozen tissue be preserved using media contain fixatives. This approach may have advantages if chemical fixation thought induce morphological artifacts or antigen redistribution.