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

1. Cut 5-6 thin strips from leaf in a cross section, avoiding major veins, approximately 1-2 mm by 3-4 mm.
2. 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
3. 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)
4. 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.
5. 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.
6. Wash 3 times in distilled water for 5 minutes each.
7. Start alcohol dehydration gradient from 15%, each wash for 10 mins, ~ 2 mLs. Continue through 30%, 50%, 70-75%, 80%, 90%, 95%.
8. Three washes at 100% Ethanol.
9. One wash at 100% Acetone.
10. 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.
11. 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.
12. 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.