by Isabel D’Alessandro

**Dissections **#

Here are some videos which show how to dissect the whole fly brain, with slight variations in technique: 

• Janelia Adult Brain Dissection 
• JoVE (1:13) 
• JoVE (3:21) 

I usually perform dissections in 3-well glass plates in saline. Following each dissection, I use a pipette to transfer the brain to a 1.5mL eppendorf tube containing 200uL PBS. After all dissections are complete, I add 24uL PFA to each tube to fix the brains. For batch processing of brains of the same genotype, I add 3-5 brains to a single tube. 

In addition to above steps, there is a more detailed protocol on Google Drive that can help you with dissections. It is linked here.

Extra note on brain+VNC dissection#

We use a small gel-filled dish and insect pins to assist brain+VNC dissection.

• Step 1: instead of using the 3-well glass plate, use the gel-filled dish. Dip the fly in 70% ethanol and then transfer the fly onto the dish.
• Step 2: orient the fly ventral side facing up, and then stick the pin to the lower part of its body. Now the fly’s position should be stably fixed.
• Step 3: add several drops of saline.
• Step 4: dissect the VNC first. A trick is to approach laterally, which avoids damaging the VNC which is very ventral and medial. Video in the above Janelia Adult Brain Dissection guide did a really good job exposing the VNC, so follow them.
• Step 5: after VNC, dissect the brain as normal procedure. Then, transfer the associated brain+VNC into one vial. Ideally one vial contains only one brain+VNC. Proceed to fixation.

Immunostaining #

The standard immunostaining protocol is described in this document. Antibody locations, dilutions, and additional information can be found in the Wilson Lab Immunohistochemistry Reagents Log. Update this spreadsheet as aliquots are added to or removed from boxes. Antibodies are generally stored in a box in the -20C lab fridge. Additional aliquots are kept in the -80C freezer in the equipment room.