Embedding Drosophila Brain in a Matrix for Time-Lapse Imaging of the Circadian Clock Neurons

Begin with a Petri dish lid containing the Drosophila larval brain, pre-exposed to a light/dark cycle. The slice contains fluorescent protein-expressing clock neurons. 

Add a nutrient-rich medium with antibiotics to support neuronal survival and a warm fibrinogen solution. 

Gently mix the solution to distribute fibrinogen evenly. 

Place a small amount of this mixture onto the coverslip of a glass-bottom dish. 

Add thrombin to react with fibrinogen to induce the formation of a three-dimensional fibrin matrix.

Transfer the brain onto this matrix, positioning it anterior-side down to align clock neurons for imaging. 

Fold the matrix over the brain to anchor it and prevent movement. 

Add nutrient-rich medium to inactivate thrombin activity. 

Seal the dish with a gas-permeable membrane to maintain hydration and neuronal viability. 

Perform time-lapse fluorescence imaging at specific intervals. 

The fluorescence changes in clock neurons reveal their rhythmic activation and inhibition, synchronized with the light/dark cycle, reflecting circadian rhythms, the daily activity cycle.

After transferring the brain into working solution, dispense the brain and medium onto the lid of a sterile petri dish. Then add another 3.5 microliters of SAM with antibiotics, and add 3.5 microliters of warm SAM with fibrinogen.

It is important to maintain the SAM medium with fibrinogen at 37 degrees throughout this procedure.

Next, mix the solution around the brain by gently pipetting with the same pipette tip and aspirate 2 microliters of the solution to deposit on a coverslip for a glass bottomed dish. Then, add 0.8 microliters of thrombin to the droplet and mix very quickly to initialize the polymerization, which will appear white. Then, use forceps to gently pick the brain and set it onto the polymerizing fibrin matrix.

Adding the thrombin after the brain was transferred to the droplet. It's not recommended as it complicates a lot the procedure.

Orient the brain as needed and very gently push the brain as close to the coverslip as possible. Then pick a layer of polymerized fibrin and fold it on top of the brain, using small and gentle movements that do not detach the matrix. Continue to fold layers of polymerized fibrin over the brain from different parts of the clot to stabilize the brain. Then deposit 20 microliters of SAM with antibiotics over the embedded brain to halt the thrombin activity. Then lay a piece of PTFE membrane over the medium and stick the edges of the membrane to the greased parts of the dish.