Materials required

0.2M Pipes buffer

Add 6 grams of Pipes to 50mls distilled water. Add N Sodium Hydroxide dropwise until solid dissolves then adjust to pH 6.8. Make up to 100mls

0.1M Sodium Cacodylate buffer

Dissolve 2.15 grams in 100mls distilled water. Adjust pH to 6.8 with N HCL

5% Glutaraldehyde - Prepare fresh before use

Add 5mls of 25% Glutaraldehyde to 10mls of 0.2M PIPES buffer. Adjust pH to 8.0 with N NaOH
And make up to 25mls with distilled water.

0.2% Osmium Tetroxide - Prepare fresh before use

Add 1ml of 2% aqueous Osmium stock solution to 9 mls of 0.1M Sodium Cacodylate buffer

Uranyl Acetate - Takes about 48 hours to dissolve

Dissolve 5 grams of Uranyl Acetate in 100mls of distilled water.

Araldite - Special Recipe

 Araldite CY212 Resin 175mls
 DDSA  275mls
 Dibutyl phthalate  5mls

Warm ingredients to 37 C, mix thoroughly and store in dark bottle at room temperature. ( Stable for about 1 year). Measure out the required volume of resin and add 1% BDMA accelerator (v/v) and mix for about 2 hours on a mixer before use. Warming the resin first assists mixing.

TEM PROTOCOL

  1. Samples were fixed in 5% Glutaraldehyde in PIPES buffer pH8.0 for 16-18 hours
  2. Buffer wash with 0.2M PIPES buffer 2 x 1 hour
  3. Then wash with 0.1M Sodium Cacodylate buffer 1 x 1 hour
  4. Post-fixed in 0.2% Osmium Tetroxide in Sodium Cacodylate for 16-18 hours
  5. Wash 2 x 30 mins in Distilled water
  6. (To help orientation of specimens, the samples were embedded in 1% Agar. When set the agar was cut into blocks containing the specimens).
  7. En-bloc stain with 5% Uranyl Acetate for 16-18 hours
  8. Wash 2 x 30 mins in Distilled water
  9. Dehydrate in 100% Ethanol for 2 x 30 mins
  10. Propylene Oxide 1 x 30 mins
  11. Infiltrate for 24-48 hrs in 1:1 propylene oxide/Araldite mixture
  12. Fresh Araldite for a further 24-72 hrs
  13. Samples were then embedded and polymerised for 72 hrs at 68-70 C
  14. Blocks were removed from oven and left for at least 24 hours before cutting

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