Sample Protocol for TRANSMISSION ELECTRON MICROSCOPY
Tissue is removed by dissection as rapidly as possible, and then cut into smaller pieces for fixation, the actual size of piece depends on the nature of the fixative used and the density of the tissue. For primary fixation with aldehyde fixatives the pieces should be no larger than 2 mm3. If tissue is larger, then smaller cubes 1mm3 can be cut before secondary fixation with osmium tetroxide. The fixative should be at least 10 times greater in volume than the specimen, also tissue samples should never be allowed to dry.
1 - Primary fixation for 1 - 4 hr at 4oC in 2.5% glutaraldehyde in 0.1M buffer pH 7.4
2 - Wash for 2 hr (or overnight) at 4oC in 3 changes of 0.1M buffer pH 7.4
3 - Secondary fixation for 1 hr at room temp in 1% osmium tetroxide in distilled water.
4 - Wash for 2 x 5 mins with distilled water
5 - Dehydration.
| 50% ethanol | 15 min |
| 70% ethanol | 15 min |
| 90% ethanol | 15 min |
| 95% ethanol | 15 min |
| 100% ethanol | 15 min |
| 100% ethanol | 15 min |
| 100% ethanol | 15 min |
| 100% ethanol | 15 min |
6 - Propylene oxide 2 x 10 min
Toxic and Flammable. Wear gloves and use in fume cupboard. DO NOT POUR DOWN SINK.
7 - Propylene oxide : Resin (1:1 mixture) 1 - 2 hours
Make up resin in plastic disposable beakers. Wear gloves.
8 - Fresh resin overnight on rotary mixer at 5rpm.
9 - Embed in mould and polymerise in oven at 60oC for 24 hours.