Protocol
Author
Jasper Pengelly
Overview
Embedding leaf sections for light/electron microscopy
Materials/Equipment
- Fume hood
- Vacuum equipment
- Glass scintillation vials
- Glass Pasteur pipettes
- Embedding mould
- 60∘C oven
Reagents
- Glutaraldehyde (hazardous)
- Formaldehyde (hazardous)
- Sucrose
- Na Cacodylate (hazardous)
- Osmium tetroxide (OsO4) (hazardous)
- 100% Ethanol
- 100% Acetone
- Araldite 502 embedding resin (standard mix)
Procedure
- Cut 5-6 thin strips from leaf in a cross section, avoiding major veins, approximately 1-2 mm by 3-4 mm.
- Put sections into scintillation vials with 3-5 mLs of buffer containing:
- 2.5 % Glutaraldehyde
- 3.5 % Formaldehyde
- 0.1-0.2 M Na Cacodylate
- ~ 0.12 M Sucrose
- Leave in vacuum overnight with lids open, leaf sections may float – but try to cover them in the buffer or with some bits of filter paper, or add 0.01% detergent (triton)
- Remove buffer with pasture pipette to appropriate waste (avoid touching leaf sections when adding/removing liquids to vials to reduce physical damage) and wash twice in 3-5 mLs buffer containing:
- 0.1 M Na Cacodylate,
- 0.12 M Sucrose (optional), shaking at low speed for 5-10 min.
- Wash leaf sections in 1% OsO4 (dilute in water) for 2 hours.
Note how long sections take to turn black – if this is slow, the alcohol dehydration steps need to be extended. - Wash 3 times in distilled water for 5 minutes each.
- Start alcohol dehydration gradient from 15%, each wash for 10 mins, ~ 2 mLs. Continue through 30%, 50%, 70-75%, 80%, 90%, 95%.
- Three washes at 100% Ethanol.
- One wash at 100% Acetone.
- Wash in ~ 2-3 mL Acetone containing:
- 5% Araldite 502 overnight shaking at RT.
- 10% Araldite over the day shaking at RT.
- 20% Araldite overnight shaking at RT.
- 40% Araldite over the day shaking at RT.
- 60% Araldite overnight shaking at RT.
- 80% Araldite over the day shaking at RT.
- 90% Araldite overnight shaking at RT.
- 95% Araldite over the day shaking at RT.
- Pure Araldite resin (float on top, let pieces sink) overnight shaking at RT.
- Pure Araldite resin (float on top, let pieces sink) overnight shaking at RT.
- Pure Araldite resin (float on top, let pieces sink) overnight shaking at RT.
- Take embedding mould and add a small amount of resin, position leaf sections as desired then cover over with resin and dry in 60 degree Celcius oven overnight.
- Remove from oven and leave to dry in fume hood overnight.
Health, safety & hazardous waste disposal considerations
- Glutaraldehyde (hazardous)
- Formaldehyde (hazardous)
- Na Cacodylate (hazardous)
- Osmium tetroxide (OsO4) (hazardous)
- All steps should be conducted in a fume hood wearing gloves and goggles.
- Consult individual chemical MSDS before use.
- Be aware of appropriate disposal for hazardous chemicals.