Yeast EM with LR White
See EM method for yeast cells. Notes below apply to the steps indicated.
4. Fixation. Try the following conditions:
3% formaldehyde Add 1/10 volume of 30% FA stock.
3% formaldehyde + 0.025% glutaraldehyde Add GA from 25% stock.
3% formaldehyde + 0.05% glutaraldehyde
3% formaldehyde + 0.1% glutaraldehyde
5. Fix for only 30 minutes at room temperature with gentle rotation.
[Optional: Quench fixative. Wash sample 2X with PC buffer at room temperature. Incubate for
15 minutes at room temperature with 50 mM ammonium chloride in PC buffer. Follow
quenching step with 2 PC buffer washes prior to digestion.]
6. Omit pre-treatment.
8. Monitor digestion carefully to avoid damage to fixed cells.
9-12. Skip these steps. Some antigens survive OsO4, although this is rare. Some antigens tolerate the
uranyl acetate en bloc staining, but I would omit this during the first trial.
13. Wash cells 3X with ddH2O.
14. Dehydration. Place EtOH solution over small pellet and allow to equilibrate by diffusion. If
pellet is large, dislodge from tube wall, or disperse. Do each step for 10 minutes (or more).