SAMPLE PREPARATION FOR TRANSMISSION ELECTRON MICROSCOPY
GENERAL SCHEDULE FOR ANIMAL TISSUE
| 1 | Chemical | Temperature | Time | Repetitions |
| Primary fixation | 2.5% glutaraldehyde in buffer | room or 0-4°C | 1-2 hours or microwave* | 1 |
| Wash | buffer | room or 0-4°C | 10-20 minutes | 3-5 |
| Secondary fixation | 1-4% osmium tetroxide in buffer | room or 0-4°C | 1-2 hours | 1 |
| Wash | buffer or distilled water** | room or 0-4°C | 10-20 minutes | 3-5 |
| en bloc staining (optional)*** | 0.5% uranyl acetate | 0-4°C | overnight | 1 |
| Wash after en bloc staining | distilled water | room or 0-4°C | 10-15 minutes | 2 |
| Dehydration |
25% ethanol Transition solvent if embedding resin is not miscible with ethanol |
room or 0-4°C |
10 minutes 10 minutes |
1 2 |
| Infiltration |
1 part resin/2 parts solvent |
room room room room |
15 minutes-overnight 15 minutes-overnight 15 minutes-overnight 15 minutes-1 hour |
1 |
| Embedding | Place in 100% resin in suitable container | 1 | 1 | 1 |
| Degassing (optional) | Place in vacuum desiccator or vacuum oven | room-60° C | 3-30 minutes | |
| Polymerization | 1 | 60-70° C | > 8 hours |
* See instructions for microwave fixation.
**Wash in water if en bloc staining is used.
** *If omitting en bloc staining, proceed to dehydration at end of washes.
GENERAL SCHEDULE FOR PLANT TISSUE
| 1 | Chemical | Temperature | Time | Repetitions |
| Primary fixation | 2.5% glutaraldehyde in buffer | room or 0-4°C | 2-4 hours or microwave* | 1 |
| Wash | buffer | room or 0-4°C | 30 minutes | 3-5 |
| Secondary fixation | 1-4% osmium tetroxide in buffer | room or 0-4°C | 2-4 hours | 1 |
| Wash | buffer or distilled water** | room or 0-4°C | 30 minutes | 3-5 |
| en bloc staining (optional)*** | 0.5% uranyl acetate | 0-4°C | overnight | 1 |
| Wash after en bloc staining | distilled water | room or 0-4°C | 10-15 minutes | 2 |
| Dehydration |
25% ethanol Transition solvent if embedding resin is not miscible with ethanol |
room or 0-4°C |
20 minutes
|
1 |
| Infiltration |
1 part resin/2 parts solvent |
room room room room |
1 hour-overnight 1 hour-overnight 1 hour-overnight 1 hour |
1 1 1 1 |
| Embedding | Place in 100% resin in suitable container | 1 | 1 | 11 |
| Degassing (optional) | Place in vacuum desiccator or vacuum oven | room-60° C | 3-30 minutes | |
| Polymerization | 1 | 60-70° C | > 8 hours |
* See instructions for microwave fixation.
**Wash in water if en bloc staining is used.
*** If omitting en bloc staining, proceed to dehydration at end of washes.
GENERAL SCHEDULE FOR SINGLE CELLS
| 1 | Chemical | Temperature | Time | Repetitions |
| Primary fixation | 2.5% glutaraldehyde in buffer | room or 0-4°C | 30 minutes or microwave* | 1 1 |
| Wash | buffer | room or 0-4°C | 5-10 minutes | 3-5 |
| Secondary fixation | 1-4% osmium tetroxide in buffer | room or 0-4°C | 20 minutes | 1 1 |
| Wash | buffer or distilled water** | room or 0-4°C | 5-10 minutes | 3-5 |
| en bloc staining (optional)*** | 0.5% uranyl acetate | 0-4°C | overnight | 1 |
| Wash after en bloc staining | distilled water | room or 0-4°C | 5-10 minutes | 2 |
| Dehydration |
25% ethanol Transition solvent if embedding resin is not miscible with ethanol |
room or 0-4°C |
5 minutes 5 minutes 5 minutes 5 minutes 5-10 minutes |
1 |
| Infiltration |
1 part resin/2 parts solvent |
room room room room |
15 minutes-overnight 15 minutes-overnight 15 minutes-overnight 15 minutes-1 hour |
1 1 1 1 |
| Embedding | Place in 100% resin in conical BEEM capsule with cap removed | 1 11 | 11 | |
| Centrifugation | 11 | 1 | 5 minutes | |
| Polymerization | 1 1 | 60-70° C | > 8 hours |
* See instructions for microwave fixation.
**Wash in water if en bloc staining is used.
*** If omitting en bloc staining, proceed to dehydration at end of
washes.
MICROWAVE FIXATION
FORMULA FOR SPURR'S LOW VISCOSITY RESIN
|
1
|
Firm
|
Firm/hard
|
Hard
|
|
ERL 4206
|
10 g
|
10 g
|
10 g
|
|
DER 736
|
6 g
|
5 g
|
4 g
|
|
NSA
|
26 g
|
26 g
|
26 g
|
|
DMAE
|
0.5 ml
|
0.5 ml
|
0.5 ml
|