SAMPLE PREPARATION FOR TRANSMISSION ELECTRON MICROSCOPY GENERAL SCHEDULE FOR ANIMAL TISSUE

1 Primary fixation Wash Secondary fixation Wash en bloc staining (optional)*** Wash after en bloc staining Dehydration Chemical 2.5% glutaraldehyde in buffer buffer Temperature Time Repetitions 1 3-5 1 3-5 1 2 1 1 1 1 2 2

room or 0-4C 1-2 hours or microwave* room or 0-4C 10-20 minutes

1-4% osmium tetroxide in room or 0-4C 1-2 hours buffer buffer or distilled water** room or 0-4C 10-20 minutes 0.5% uranyl acetate distilled water 25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol Transition solvent if embedding resin is not miscible with ethanol 0-4C overnight

room or 0-4C 10-15 minutes room or 0-4C 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes 10 minutes

Infiltration

1 part resin/2 parts solvent 1 part resin/1 part solvent (optional) 2 parts resin/1 part solvent 100% resin

room room room room

15 minutesovernight 15 minutesovernight 15 minutesovernight 15 minutes-1 hour 1 3-30 minutes > 8 hours

1 1 1 1 1

Embedding Degassing (optional) Polymerization

Place in 100% resin in suitable container

Place in vacuum room-60 C desiccator or vacuum oven 1 60-70 C

* 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 Primary fixation Chemical Temperature Time Repetitions 1

2.5% glutaraldehyde in buffer room or 0-4C 2-4 hours or

microwave* Wash Secondary fixation Wash en bloc staining (optional)*** Wash after en bloc staining Dehydration buffer 1-4% osmium tetroxide in buffer buffer or distilled water** 0.5% uranyl acetate distilled water 25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol Transition solvent if embedding resin is not miscible with ethanol Infiltration 1 part resin/2 parts solvent 1 part resin/1 part solvent (optional) 2 parts resin/1 part solvent 100% resin room room room room 1 hour-overnight 1 hour-overnight 1 hour-overnight 1 hour 1 3-30 minutes > 8 hours 1 1 1 1 11 room or 0-4C 30 minutes room or 0-4C 2-4 hours room or 0-4C 30 minutes 0-4C overnight 3-5 1 3-5 1 2 1 1 1 1 2

room or 0-4C 10-15 minutes room or 0-4C 20 minutes 20 minutes 20 minutes 20 minutes 30 minutes

Embedding Degassing (optional) Polymerization

Place in 100% resin in suitable 1 container Place in vacuum desiccator or room-60 C vacuum oven 1 60-70 C

* 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 Primary fixation Wash Secondary fixation Wash en bloc staining (optional)*** Chemical 2.5% glutaraldehyde in buffer buffer 1-4% osmium tetroxide in buffer buffer or distilled water** 0.5% uranyl acetate Temperature Time room or 0-4C 30 minutes or microwave* room or 0-4C 5-10 minutes room or 0-4C 20 minutes room or 0-4C 5-10 minutes 0-4C overnight Repetitions 11 3-5 11 3-5 1

Wash after en bloc staining Dehydration

distilled water 25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol Transition solvent if embedding resin is not miscible with ethanol

room or 0-4C 5-10 minutes room or 0-4C 5 minutes 5 minutes 5 minutes 5 minutes 5-10 minutes

2 1 1 1 1 2

Infiltration

1 part resin/2 parts solvent 1 part resin/1 part solvent (optional) 2 parts resin/1 part solvent 100% resin

room room room room

15 minutesovernight 15 minutesovernight 15 minutesovernight 15 minutes-1 hour 11

1 1 1 1

Embedding

Place in 100% resin in 1 11 conical BEEM capsule with cap removed 11 11 1 60-70 C

Centrifugation Polymerization

5 minutes > 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
Place glass and grid sheet in microwave with numbered coordinates aligned with left edge of plate. Place 400-ml beaker with 300 ml distilled water in back left corner of the oven. Heat on COOK 1-2 min. Replace heated beaker with fresh beaker of water (same amount and same position in microwave) Place 3 ml of sample in fixative in flat bottom glass tube. Place tube on square at X-18 coordinates. Heat 15 sec on COOK 1.

FORMULA FOR SPURR'S LOW VISCOSITY RESIN

1 Firm Firm/hard Hard

ERL 4206 DER 736 NSA DMAE

10 g 6g 26 g 0.5 ml

10 g 5g 26 g 0.5 ml

10 g 4g 26 g 0.5 ml