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ImEM of frozen section
＊download PDF| Author | Masataka Kunii | ||
|---|---|---|---|
| Affiliation | Department of Cell Biology, Graduate School of Medicine, Osaka University | ||
| Contact | inquiry about this protocol | ||
| Home Page | http://www.med.osaka-u.ac.jp/pub/acb/eng.html | ||
| Keyword | |||
| Published | 2015-08-26 | Last Update | 2015-08-26 |
| View | 18 | download PDF | 3 |
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
