The Huck Institutes of the Life Sciences

Imaging a juvenile musse

An example of imaging samples of a juvenile mussel, which were collected in the field and transported to the facility on ice
 
A juvenile Lasmigana subviridis

A juvenile Lasmigana subviridis

Samples of a juvenile mussel, Lasmigana subviridis, were fixed at the collection site with a modified Karnovsky's fixative and transported to the Electron Microscopy Facility on ice. Double-strength aqueous fixatives and buffer were carried to the collection site and freshly mixed v/v just before sampling.

Protocol

  1. Fix on site in 2.5% paraformaldehyde, 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH=7.4) and rinse in the same buffer 3 x 5 min upon arrival in the EM lab
  2. Postfix in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH=7.4) for 1 hour at rm temperature
  3. Rinse in the same buffer 3 x 5 min
  4. Dehydrate through a gradient series of ethanol (25%, 50%, 70%, 85%, 95% and 100% (x3) for 5 minutes at room Temperature
  5. Critical Point Dry the samples with bone-dry liquid carbon dioxide (4 x 3 minute exchanges + 8 minute vent)
  6. Mount the coverslips onto an aluminum stub with a double sticky tab and colloidal silver
  7. Mount onto an aluminum stub with a double sticky tab and colloidal silver
  8. Sputter-coat 10 nm of gold/palladium (2 min at 10 mAmps)