Fluorescence Staining On Cryostat Sections Of Fly Heads

  1. Embed up to 20 fly heads (10 for control flies, otherwise 15-20) in O.C.T. Tissue Tek compound (Miles Scientific) under a dissecting microscope. The heads should be oriented upright on the block, so that the ocelli are pointing towards the microscope objectives.
  2. Put samples on dry ice for 30 minutes.
  3. Place the samples, slide holder and mounts in the cryostat for 20-30 minutes prior to cutting to equilibrate them to the cryostat temperature. Cut 10 μm sections on the cryostat and transfer section ribbons onto slides.
  4. Dry slides for 10 minutes at room temperature.
  5. Fix the tissue sections in 4% paraformaldehyde in 1xPBS for 15 minutes at room temperature in a coplin jar.
  6. Wash the slides three times in PBT (1xPBS + 0.3% Triton X-100) for 10 minutes/wash.
  7. After the last wash, tap sides of slides on a paper towel to remove excess PBS and allow to air dry for 10 minutes at room temperature. (The slides may be left to dry for a few hours at this point with no apparent loss of enzyme activity.)
  8. Prepare 100 μl per slide of appropriate dilution of 1° antibody in BBT 250 (BBT + 250mM NaCl).
  9. Place slides on a level surface in a moist airtight chamber. (A tupperware box with water-soaked blotting paper works well for this purpose. To keep the slides off the paper, they should be placed on two lengths of plastic 5ml or 10ml pipettes that rest on top of the paper block.) Apply 90-100μl of 1° antibody dilution to each slide and cover with a plastic coverslip.
  10. Incubate slides for 2 hours at room temperature or overnight at 4°C.
  11. Wash slides three times in PBT for 10 minutes/wash.
  12. Allow slides to air-dry for about 10 minutes.
  13. Prepare 100μl per slide of appropriate dilution of 2° antibody in BBT 250. Place slides back on level surface of moist airtight chamber. Apply 90-100μl of 2° antibody dilution to each slide and cover with a plastic coverslip.
  14. Incubate slides for 2 hours at room temperature or overnight at 4°C.
  15. Wash slides three times in PBT for 10 minutes/wash.
  16. Mount slides in Vectashield Mounting Medium and store in slide tray.