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