Day1
Dissection:
• 3-5 days old flies are dissected in ice-cold Schneider’s Insect Medium.
• Dissected tissues are fixed in 2% PFA at 25 ̊ C for 55 min.
• Fixed tissues are washed with 0.5% PBT, 3x, 10 min each 
Dehydration:
• Dehydrate the fixed tissues with graded EtOH series: 30%, 50%, 70%, 100%, 100%, 100%, 10 min each.
• Dehydrated tissues are stored at 4 ̊C with 100% EtOH overnight on a rocker.
Day2
Permeation:
• Dehydrated tissues are rehydrated with graded EtOH series: 70%, 50%, 30%,  10 min each.
• Rehydrated tissues are incubated at 4 ̊C 5% acetic acid for 5 min \(do this in the cold room or on ice).
• Wash tissues with 4 ̊C 1xPBS, 3x, 5 min each.
• Fix tissues with 2% PFA at 25 ̊C for 55 min.
• Wash tissue with 0.5% PBT, 3x, 10 min each.
Autofluorescence quenching:
• Prepare 1xPBS with 1% sodium borohydride at 4 ̊C \(prepare fresh, use 4 ̊C 1xPBS to prepare the solution).
• Incubate tissues in 1xPBS with 1% sodium borohydride for 30 min at 4 ̊C, change solution every 10 min \(do this in the cold room or on ice).
• Wash with 4 ̊C 1xPBS for 3x, 5 min each.
Pre-Hybridization:
• Prepare fresh pre-hybr soln with formamide 
• Incubate tissues with pre-hybr soln at 50 ̊C for 2 hrs.
Hybridization:
• Incubate FISH probes in 90 ̊C for 3 min, then snap cold the FISH probes on ice for 10 min.
• Prepare ~48μL hybr soln for 1 reaction, which can stain 3-5 brains.   
• Add 1-2 μL of 50-100ng/μL of FISH probes to the hybr soln \(total reaction value is 50μL)
• Incubate at 50 ̊ C for 10 hrs, then at 37 ̊ C for 10 hrs. Day3
Washing:
• Warm up the pre-hybr soln to 37 ̊C.
• Add 200μL 37 ̊C pre-hybr soln directly to the hybridization reaction, incubate for 10 min.
• Wash with pre-hybr soln, 1x, 37 ̊C, 10 min. 
• Wash with pre-hybr soln, 1x, 25 ̊C, 10 min. 
• Wash with 30% formamide washing soln, 2x, 25 ̊C, 30 min each .
• Wash with 2xSSC washing soln, 3x, at 25 ̊C, 10 min each 
• Wash with 1xPBS, 1x, at 25 ̊C, 10 min 
• Fix tissues at 2% PFA at 25 ̊C for 55 min.
• Wash with 0.5% PBT, 3x, at 25 ̊C, 10 min each.
Clearing and mounting:
• Mount tissues on glass coated with poly-L-lysine.
• Dehydrate the tissues with graded EtOH series: 30%, 50%, 75%, 100%, 100%, 100%, 10 min each.
• Clear tissues in xylene, 3x, 5 min each \(perform this step in a fume hood)
• For confocal imaging, mount tissues in DPX \(Electron microscopy sciences).
• For BB-SIM, hold tissues in xylene until imaging.