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Imaging insect blood cells

Imaging insect blood cells

Manduca sexta (inset) blood cell attached to a coverslip coated with 0.5% laminarin. Vertical bar (bottom left) = 1 µm.

Manduca sexta larvaTobacco hornworm (Manduca sexta) is a moth whose larvae feed on a variety of plants in the family Solanaceae. Its large size and short life cycle can make it a useful study organism.

Here we were examining Manduca sexta's blood cells, using TEM.

Protocol

  1. Manduca sexta bled onto permanox coverslips in tissue culture plates in presence on 0.5% laminarin; incubate at room temperature overnight
  2. Add 1.5% glutaraldehyde, 2.5 % formaldehyde in 0.1 M phosphate buffer, pH 7.2 with 4% sucrose to plates, incubate 2 hr at room temperature
  3. Wash 3 times in 0.1 M phosphate buffer
  4. 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2, incubate 1 hr at rm temp
  5. Wash 1 time in 0.1 M phosphate buffer, 2 times in water
  6. En bloc stain 1 hr in 2% aqueous uranyl acetate
  7. Dehydrate in ethanol, replace ethanol with acetone, then infiltate with Spurr's resin
  8. Polymerize 24 hrs at 60C
  9. Ultrathin sections are cut and placed on uncoated copper grids
  10. Contrast with uranyl acetate and lead citrate