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TEM Of Botanical Samples

(Soybean Root Nodules)

Protocol for Mary Lou Guerinot's lab, Biology Dept.

References: Jelesko J G et al. 1993, MPMI 6(1), 135-143

Vasse J. et al. 1990, J. Bacteriol. 172(8), 4295-4306

Stock buffers:

0.4 M Na Cacodylate + 5 mM CaCl2 pH 7.2

0.3 M Na Cacodylate + 2.5mM CaCl2 pH 7.2

TEM Protocol:

1. Fix in 3% GTA/1%PF in 0.2M NaCac buffer with 2.5 mM CaCl2 pH7.2 :

Cut 1-3mm3 root nodule discs and place in fresh fix; apply gentle vacuum until specimens sink; then complete fine trimming of samples(@1mm3), if necessary. Place in fresh fix and leave under vacuum overnight.

Fix prep: 2ml 70% GTA + 11ml. 0.4 M Cacodylate with 5 mM CaCl2, pH7.2; 22ml. 0.2 M Cacodylate with 2.5 mM CaCl2, pH7.2 and 11ml 4%PF in dH2O with 5mM CaCl2.

DAY 1

2. Rinse in 0.3M NaCac buffer with 2.5mM CaCl2, pH7.2. several washes over one hour, with light vacuum

3. Post-fix in 2% OsO4 in 0.25M NaCac buffer with 2.5mM CaCl2, pH7.2, for 3 hours at RT.

Fix Prep: 1 vol. 4% OsO4 in dH2O. + 1 vol. 0.4 M Cacodylate with 5 mM CaCl2, pH7.2 + 2 vol. 0.3 M Cacodylate with 2.5 mM CaCl2, pH7.2.

4. Rinse in dH2O. Dehydrate 30%, 50% 70% 95% ETOH 15-30 minutes each.

5. Rinse in 100% ETOH three times over 1 hour. Can store in fridge at this point, if necessary.

DAY 2

6. Two changes in Propylene Oxide 15-30 minutes each.

EPON:PO {1.5:1} ? several changes over 3-4 hours in sealed vials, on rotator; then under vacuum overnight.

DAY 3

7. Transfer to BEEM Capsules and fresh EPON under vacuum for 24 hrs:

DAY 4

8. Polymerize(to enhance infiltration of the tissue block:

a. Heat 8 hrs. at 45 C.

b. Increase heat to 60 C for and additional 40 hrs.

Last Updated: 10/2/08