List of abbreviations |
Vocabulary
of micros- copic anatomy specialist terms explained in English + German |
Every attempt was made to provide correct information, however any liability for eventual errors in the protocol is rejected! |
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Conditions of use |
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