specimen preparation for transmission electron microscopy Dr. Jastrow's electron microscopic atlas

Vocabulary
of micros-
copic
anatomy
specialist terms
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German

Every attempt was made to provide correct information, however any liability for eventual errors in the protocol is rejected!

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Editor:
Dr. med.
H. Jastrow

Conditions
of use

specimen preparation for transmission electron microscopy
Preface:
The metod descibed here is an example for fixation, contrastation and embedding only. It worked well for retina and pineal gland tissue.
For optimal results especially the fixation needs to be adapted to the investigated tissue.
It is of major importance that the fixing agent is able to completely! penetrate the specimen as soon as possible. For this reason only very small peaces of tissue (~ 1 mm³) should be fixed if no perfusion fixation (fixative is directly infused into a larger vessel of a deeply anaesthized animal) should be possible. The preparation should be quick enough, i.e. 5 minutes after tissue does no longer recieve oxigen, it begind to show first signs of degeneration of ultrastructures.

Method:
- Perfusion or immersion fixation of the tissue using a modified Karnovsky (1965) sloution:
2.5 % buffered glutaraldehyde + 2 % paraformaldehyde in 0.1 M sodiumphosphate buffer (Sörensenbuffer) pH 7.4
- leave tissue overnight at 4° C
- wash 3 x 15 minutes (min.) in 0.1 M sodiumphosphate buffer + 0.1 M Sucrose
- postfix 90 min. in 2 % sodiumphosphate buffered osmiumtetroxide pH 7.4
- wash 3 x 15 min in 0.1 M sodiumphosphate buffer pH 7.4
- dehydrate 2 x 15 min: 50 % aceton (in destilled water)
- contrast overnight using 70 % aceton + 0.5 % uranylacetate + 1 % phosphotungstic acid at 4° C
- 2 x 15 min. 80 % aceton
- 2 x 15 min. 90 % aceton
- 2 x 15 min. 96 % aceton
- 3 x 20 min. 100 % aceton
- 2 x 15 min. propylenoxyde
- 30 min. 2 : 1 propylenoxyde : Epon mixture
- 30 min. 1 : 1 propylenoxyde : Epon mixture
- 30 min. 1 : 2 propylenoxyde : Epon mixture
- Epon solution overnight at 4° C
- new fresh Epon solution
- put in incubator for ~48 hours at 65° C for polymerisation
- cut with an ultramicrotome set to 50 - 100 nm section thickness
- rinse sections to grids or gelatine-covered one-whole grids made of cooper or nickel
- postcontrastation of sections according to Reynolds (1963):
- 10 min. 8 % uranylacetate
- 5 min 0.7 % leadcitrate + 0.9 % sodiumcitrate
- after drying for ~15 min sections may be investigated in a transmission electron microscope

Literature:
Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212.
Karnovsky, M.J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137-138.

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