Biochemistry Lab
MAKE SURE CELLS ARE IN POLYPROPYLENE TUBES, NOT POLYSTYRENE
1. Yeast cells in media. Dilute 1:1 with stock 8% GTA in H20. Concentration of fix is 4% in 50 % media. After 5 minutes centrifuge(slow-clinical) and replace with 4% GTA in 0.1 M NaCacodylate(NaCac) pH 7.4 for 2 hrs.
2. Rinse twice in 0.1 M NaCac. pH 7.4
3. Post-fix in 2%OsO4 in dH20 for 1 hour room temp.
4. Rinse in dH20.
5. Dehydrate thru ETOH 30%, 50 %, 70 %, 95 %, 5 mins. each.
6. 100 % ETOH 3X, 5 minutes each.
7. Propylene Oxide(PO) 2X, 5 minutes each.
8. LX112 epoxy:PO 1.5: 1 ; capped for 2-3 hrs. Because of poor infiltration, keep a vacuum as long as possible; then vacuum overnight in desiccator.
9. Cells into BEEM capsules; Fresh EPON; Because of poor infiltration; keep under vacuum for at least 8 hrs.; then overnight in vacuum desiccator.
10. Polymerize for 48 hours at 70oC.
Section Stain: 2% UA aqueous 45-50' LC 3' or 2% UA in MEOH 20', LC 5'
TEM PREP FOR YEAST CELLS WITH CELL WALL REMOVAL
Byers, etal ,1991, "Preparation of Yeast Cells for TEM", Methods in Enzymology Vol 194 pp. 601-627.
1. Yeast cells in media. Dilute 1:1 with stock 8% GTA in H20. Concentration of fix is 4% in 50 % media. After 5 mins. Transfer cells to fresh GTA Fix: 2% GTA in 40mM phosphate-magnesium buffer(40mM K2HPO4-KH2PO4 pH6.5, 0.5mM MgCl2). Fix cells in suspension for 30 minutes. At this point, the cells can be stored overnight at 2oC.
2. Rinse twice in 0.1 M Phosphate-citrate buffer, pH 5.8
0.2 M Phosphate-citrate buffer:0.17M K2HPO4 and 30mM NaCitrate to yield pH 5.8
Dilute 1:1 w dH20
3. Resuspend in 0.1 M Phosphate-citrate buffer, pH 5.8, containing a 1/10 dilution of Glusulase. Incubate (@ 2hrs.) until walls have been removed, as indicated by loss of wall refractility under phase-contrast microscopy.
4. Rinse twice in 0.1 M NaAcetate buffer, pH 6.1(adjust pH with Glacial acetic acid).
5. Post-fix in 2%OsO4 in 0.1 M NaAcetate buffer, pH 6.1, for 15 mins. room temp.
6. Rinse in dH20.
7. Incubate in 1% aqueous UA(in the dark) 60 mins.
8. Rinse in dH20.
9. Dehydrate thru ETOH 30%, 50 %, 70 %, 95 %, 5 mins. each.
10. 100 % ETOH 3X, 5 minutes each.
11. Propylene Oxide(PO) 2X, 5 minutes each.
12. LX112 epoxy:PO 1.5: 1 ; capped for 2-3 hrs. Because of poor infiltration, keep a vacuum as long as possible; then vacuum overnight in desiccator.
13. Cells into BEEM capsules; Fresh EPON; Because of poor infiltration; keep under vacuum for at least 8 hrs.; then overnight in vacuum desiccator.
14. Polymerize for 48 hours at 70oC.
Section Stain: 2% UA aqueous 45-50' LC 3' or 2% UA in MEOH 20', LC 5'
