#  SEM Preparation 

 



##  SEM Preparation 

 

 

       ![SEM of Squid Tentacle Sucker](/sites/g/files/omnuum4896/files/styles/hwp_21_9__1920x825/public/electron-microscopy/files/squi_tentacle_sucker.jpg?itok=mL9BOxgT) 

 

 



 

 



 

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1. **Fixation** Fixation 1-2 hrs (or overnight at 4C) 2.5% Glutaraldehyde 2.5% Paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4).
2. **Rinse** 3 X 10 min in 0.1M sodium cacodylate buffer, pH 7.4
3. **Post-Fix** 1-2 hrs in 1% Osmium tetroxide in water
4. **Rinse** 3 X 5 min in water
5. **Dehydrate**  
    50% ETOH 5 min  
    70% ETOH 5 min  
    95% ETOH 5 min  
    100% ETOH 10 min  
    100% ETOH 10 min
6. **Drying:** Critical Point Dry (CPD) or dry using HMDS.
7. **Mount specimens** onto stubs by using conductive carbon tape, silver paint, or colloidal graphite. If you use paint or graphite, it must be dried overnight before going into the Sputter Coater.
8. **Sputter Coating.** The thickness you use can vary between 1- 10 nanometers.
9. **SEM Imaging**



 

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 Attachments- [  picture\_as\_pdf  Routine Protocol for Biological SEM.pdf ](/sites/g/files/omnuum4896/files/2024-11/Routine%20Protocol%20for%20Biological%20SEM.pdf)
 
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