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