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.