日本細胞生物学会Japan Society for Cell Biology

ImEM of frozen section

AuthorMasataka Kunii
AffiliationDepartment of Cell Biology, Graduate School of Medicine, Osaka University
Contactinquiry about this protocol
Home Pagehttp://www.med.osaka-u.ac.jp/pub/acb/eng.html
Keyword
Published2015-08-26Last Update2015-08-26
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Summary

Methods

Detail  *Click photo to see a large size.

    • PBS
      5%NDS/PBS
      Diluted primary antibody in 5%NDS/PBS, 37℃ 1hr
      PBS
      5%NDS/PBS
    • Diluted secondary antibody (Fluoronanogold 1/20) in 5%NDS/PBS RT 1hr
    • PBS x 3 and check the fluorescence under light microscopy
    • If you can see specific staining, fix the sections with 1%GA/PBS, RT 10min
    • DDW x 3
      Silver Enhance(A+B+C) RT 5-20min depending on the temperature and efficiency of primary antibody *usually 10min x 1 + 5min x 1
      DDW x 3
    • Selenium toner (KODAK) 1/20 RT 7min to prevent erosion of silver during OsO4 fixation
    • DDW x 3
      Postfix with 2%GA/0.1M Cacodylate Buffer (CB) (pH7.2) RT 5min
    • 10%Suc/0.1M CB 4℃ 5min
      1% OsO4/0.1M CB 4℃ 20min
      DDW x 3
    • 0.5% uranium acetate 4℃ O/N or 1% uranium acetate RT 20min
    • Dehydration 65% EtOH to 100% EtOH 5min/each step
    • Absolute EtOH RT 10min×3 Propylene oxide RT 10min×2 Epon:Propylene oxide RT 5min
    • Pour epon on the slide glass and remove it shortly×3
    • Put epon cylinder on the slide glass and put it into the oven to polymerize the epon 65℃, more than 36hrs
    • After polymerization, remove a part of epon on the slide with a razor blade and put the slide glass with an epon cylinder on a hot plate at about 150℃. Wait for about 20seconds to make the epon soft.
    • Push over the epon cylinder and the section will be peeled off from the slide

Little trick

Reference