5.6.1. Preparation and embedding of larval tissues

  1. Perform infection assays as described in section 4.1.
  2. Wash each larva for 5 seconds with 100 % ethanol.
  3. Fix larvae by overnight incubation with 4 % Roti Histofix (Roth, Karlsruhe) at 4°C at 20 rpm.
  4. Wash samples overnight with 1X PBS solution containing 6.8 % sucrose under the same conditions.
  5. Wash samples with 100 % acetone.
    During the first five minutes, change acetone until the solution becomes clear.
  6. Incubate with the final change of acetone for 1 hour under the same conditions.
  7. Mix equal volumes of 100 % acetone and Technovit 8100 solution (Heraeus Kulzer, Wehrheim).
  8. Incubate samples for 2 hours with this solution under previous conditions.
  9. Perform infiltration and embedding using Technovit 8100 according to manufacturer´s instructions.
  10. Cut 4 µm-sections using a microtome, straighten them with warm sterile water on slides.
  11. Store slides at 4°C until further use.