9.2. Tissue fixation and tissue sectioning exemplified with gut tissue

  1. Immobilize about 20 bees withCO2.
  2. Cut of the head.
  3. Fix the abdomen on a separation plate with micro pins.
  4. Remove carefully the alimentary tract of each bee with forceps.
  5. Transfer the hindgut and the midgut into one well of a 24-well microtiter plate.
  6. Fix tissues in 4% formalin (Roth) for 24 hours at 4°C by shaking.
    The further embedding and blocking procedure using e.g. Technovit 8100 and Technovit 3040 kits (Heraeus-Kulzer) should be performed as given in the manufacturer´s protocols (Heraeus-Kulzer, T8100 embedding kit).
  7. Wash the alimentary tracts with 6.8% sucrose in 1xphosphate buffered saline (1xPBS, pH 7.0) for 24 hours at 4°C.
  8. For dehydration transfer the tissue in 100% acetone for one hour.
  9. Pre-infiltrate the organs with T8100 basic-solution and 100% acetone (mixed 1:1) for two hours.
  10. Prepare the infiltration solution (0.6 g hardener I in 100 ml T8100 basic solution).
  11. Transfer the organs into the infiltration solution.
  12. Incubate at 4°C for at least 24 hours and up to one week, depending on tissue size.
  13. Apply careful shaking for better infiltration results.
  14. Prepare the embedding solution (mix 0.5 ml hardener II with 15 ml infiltrating solution).
  15. Fill the mold of a Teflon-embedding form (pre-cooled at -20°C) with the embedding solution.
  16. Transfer the tissue and orientate it in the mold.
  17. Close the well immediately with a plastic strip.
  18. Incubate at 4°C for 3 hours to allow polymerization.
  19. Finally, block the polymerized probes with histoblocs and with the Technovit 3040 kit (both from Heraeus-Kulzer) using the manufacturer´s protocol.
  20. Prepare semi thin-sections (2-4 µm) with a rotation microtome (Leica).
    Use a knife with a hard metal edge (Tungsten).
  21. For fluorescence in situ-hybridization transfer the tissue sections on Polysine™-covered glass slides (Fisher Scientific, Menzel-Gläser).