Standard SEM Protocol
This protocol was contributed by Kirsten Bomblies and Vipula Shukla (Salk Institute) and Charles Graham (Scripps Institution of Oceanography).
FAA fixative 50% ethanol 5% (v/v) acetic acid 3.7% (v/v) formaldehyde
Osmium tetroxide (0s04) (1% [v/v] 0s04 in 25 mm sodium phosphate buffer at pH 7.2)
Store the solution wrapped in aluminum foil at 4°C. The solution will keep for at least 1 month; discard it when it turns purple. OsO is very toxic and should always be handled with great caution, useci in a chemical fume hood, and disposed of in appropriate chemical waste containers.
Ethanol (100%, stored over molecular sieves)
Equipment for SEM
CAUTION: acetic acid, C02, ethanol, formaldehyde, osmium tetroxide (see Appendix 3)
1. Fix tissue by harvesting and placing directly into fixative solution in glass scintillation vials.
2. Briefly apply a vacuum to remove air bubbles from the tissue; release vacuum slowly.
3. Incubate in fixative for 12-24 hours at 4°C.
4. Replace fixative with just enough 0s04 solution to cover the samples.
5. Incubate at 4°C overnight or longer (up to a few days). The tissue should turn black (some tissue types will remain gray).
6. Rinse the samples three times in 25 mm sodium phosphate at pH 7.2.
7. Dehydrate through an ethanol series at least 15-30 minutes at room temperature through 30%, 50%, 70%, 95%, 2x 100% ethanol. Store tissue in 100% ethanol until continuing with critical point drying.
8. Incubate the samples overnight in 100% ethanol. Place the samples into baskets, which are in the boat (part of the specimen holder assembly of the critical-point-drying apparatus) filled with 100% ethanol. Cover the baskets with wire mesh to hold them in place.
9. Use a pipette to remove most of the ethanol from the boat and transfer the boat immediately into the critical-point-drying apparatus. Tighten the seal with an Allen wrench.
10. Open the first valve on the C02 tank and then the inlet valve of the drying apparatus.
11. Relieve the back pressure by opening the vent, until the C02 fills the chamber.
12. Close the inlet valve and open the drain at the bottom of the dryer to remove ethanol.
13. Repeat Steps 11 and 12.
14. Close the vent and let the C02 sit in the chamber for at least 10 minutes.
15. Change C02 six times as follows:
a. Open the drain and allow the C02 level to drop all the way.
b. Open the inlet valve and relieve back pressure by opening the vent, allowing the C02 level to rise to the top of the chamber again.
c. Close drain, inlet, and vent and allow the C02 to sit for at least 10 minutes.
16. To reach the critical point, drain C02 half way. A meniscus should be clearly visible. Close all of the valves, including the main tank valve.
17. Turn on the hot water inlet to the drying apparatus, watching the meniscus and gauges as the temperature and pressure approach the critical point (1150 psi, 32°C). Once the meniscus disappears, immediately turn off the hot water.
18. Leave samples at the critical point for 5 minutes.
19. Slowly (over at least 5 minutes) bleed through the vent in a steady flow. Open the vent further as pressure drops.
20. Store the samples in a dry container until ready for mounting.
21. Mount the samples on stubs using sticky tabs (or double-sided mounting tape) and silver colloid. For small Arabidopsis samples, place a droplet of colloid (not too fluid) on the surface of the stub, and then stick the sample into colloid with fine forceps. Gently support the sample until the colloid begins to set. At this point, allow the colloid to dry for -1 hour if further dissection of sample is desired.
22. Dissect samples as needed with microdissection tools.
Homemade tools can be fashioned by drawing out glass pipettes or fixing eyelashes to toothpicks.
23. Coat samples with gold and palladium according to the instructions for the sputter coater. Examine the coated samples under SEM or store them in boxes (to prevent damage and dust accumulation) until ready to view.
If further dissection is required after viewing, the samples must be recoated.
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