Fixation for SEM

SAMPLE PREPARATION FOR SCANNING ELECTRON MICROSCOPY

GENERAL SCHEDULE FOR ANIMAL TISSUE

1	Chemical	Temperature	Time	Repetitions
Primary fixation	2.5% glutaraldehyde in distilled water	room or 0-4°C	1-2 hours or microwave*	1
Wash	distilled water	room or 0-4°C	10-20 minutes	3-5
Secondary fixation	1-4% osmium tetroxide in distilled water	room or 0-4°C	1-2 hours	1
Wash	distilled water	room or 0-4°C	10-20 minutes	3-5
Dehydration	25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol	room or 0-4°C	10 minutes 10 minutes 10 minutes 10 minutes 10 minutes	1 1 1 1 2
Critical point dry
Mount on specimen stub with silver paste or graphite
Coat with gold/palladium alloy
Store stubs in desiccator

* See instructions for microwave fixation.

GENERAL SCHEDULE FOR PLANT TISSUE

1	Chemical	Temperature	Time	Repetitions
Primary fixation	2.5% glutaraldehyde in distilled water	room or 0-4°C	2-4 hours or microwave*	1
Wash	distilled water	room or 0-4°C	30 minutes	3-5
Secondary fixation	1-4% osmium tetroxide in distilled water	room or 0-4°C	2-4 hours	1
Wash	distilled water	room or 0-4°C	30 minutes	3-5
Dehydration	25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol	room or 0-4°C	20 minutes 20 minutes 20 minutes 20 minutes 30 minutes	1 1 1 1 2
Critical point dry
Mount on specimen stub with silver paste or graphite
Coat with gold/palladium alloy
Store stubs in desiccator

* See instructions for microwave fixation.

GENERAL SCHEDULE FOR SINGLE CELLS

1	Chemical	Temperature	Time	Repetitions
Primary fixation	2.5% glutaraldehyde in distilled water	room or 0-4°C	30 minutes or microwave*	1
Wash	distilled water	room or 0-4°C	5-10 minutes	3-5
Secondary fixation	1-4% osmium tetroxide in distilled water	room or 0-4°C	20 minutes	1
Wash	distilled water	room or 0-4°C	5-10 minutes	3-5
Dehydration	25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol	room or 0-4°C	5 minutes 5 minutes 5 minutes 5 minutes 5-10 minutes	1 1 1 1 2
Place porous container in small amount of 100% ethanol in petri dish Place specimen in porous container Place lid on container. If drying more than one sample, mark with notch (s) to identify sample
Critical point dry
Place double-stick tape on specimen stub
Sprinkle specimens onto tape, Dust with camel hair brush or other implement to spread sample
Coat with gold/palladium alloy
Store stubs in desiccator

* See instructions for microwave fixation.

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.