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.