General Drosophila Whole-Mount Histochemical Staining Method  

Richard Carthew 

 

High-Octane Fix:         3.38 ml 37% formaldehyde

                                    0.5 ml 1M Na2PO4 pH6.8 buffer

                                    1.12 ml water

                                    5 ml octane (Sigma)

                                    Vortex 60 seconds.  Let settle and use upper octane phase only.

 

 

Post-Fix:                     0.43 ml 37% formaldehyde

                                    0.4 ml 1M Na2PO4 pH6.8 buffer

                                    3.17 ml water

                                    Mix and chill on ice.

 

 

  1. Place Drosophila (larvae, pupae or adults) into High-Octane Fix at RT and fix for 20 minutes.

 

  1. Remove Fix and wash once with octane.  Remove octane and replace with PBS.

 

  1. Dissect open flies to expose underlying tissues. Adult heads, dissect from bodies and remove proboscis.

 

  1. Replace PBS with Post-Fix and fix for 60-90 minutes on ice. 

 

  1. Wash twice with PBS and complete tissue dissection of specimens while they are in PBS.  For adult heads, use Dumont forceps to peel off head cuticle.  Retinas with attached laminae can also be peeled from brain.  For thoracic ganglia, make a mid-sagittal slit along dorsal thorax and dissect away muscle groups to reveal a ventral ganglion (white) near the legs.  Nudge the ganglion from its place.

 

  1. Incubate in PBS + 0.3% Triton for 30 minutes at RT.

 

  1. Wash twice for 10 minutes each in PBST (PBS + 0.1% Triton).

 

  1. Incubate 30 minutes in PBST + 10% goat serum.

 

  1. Incubate 18 hours at 4¡C in primary antibody diluted in PBST + 10% goat serum.

 

  1. Wash four times, 30 minutes each in PBST at RT.

 

  1. Incubate 2-3 hours at RT in fluorescent secondary antibody in PBST + 5% goat serum.

 

  1. Wash four times, 30 minutes each in PBST, and once in PBS at RT.

 

  1. Transfer to slide, wick dry PBS and replace with a drop of AntiFade: 90% glycerol, 10mM Na2CO3 pH9.6, 100mg/ml 1,4-diazabicyclo[2.2.2]-octane (Aldrich).  Cover slip.