Neurodegeneration Assay for Dopaminergic Reporter Strains

Embryos were obtained by hypochlorite treatment of gravid adults from different genotypes of worms. After 17–24 h incubation in M9 buffer to obtain synchronized L1s, the worms were washed once in 10 ml dH₂O, spread on OP50-1 containing plates and incubated at 20°C during the whole assay time period. The assay was conducted at different stages of worms’ life cycle : L4 larvae, day 5 and day 10 adults.

On the day of the microscopy worms are immobilized with 3mM levamisole on 4% agarose pads and were scored immediately.
For quantitative analyses of dopaminergic neurodegenration, approximately 50 worms were examined under a stereofluorescent Compound microscope (Olympus, Hamburg, Germany) using filters for excitation wavelengths from 455 to 490 nm at 60X Oil objective.

For dopamine neuron analyses using the DAT-1 promotor, all four cephalic sensilla (CEP) dendrites were examined and GFP fluorescence within the dendrites followed from the nerve ring to the tip of the nose along with the two anterior dierdic (ADE) neurons. 
If any part of the dendrite was absent, the worm was considered to have altered dopaminergic neurons and was scored as positive for dopamine neural loss. Other phenotypes such as axonal blebbing (broken neurites) & axonal branching / outgrowth were counted likewise.

All the worms that have any of these defects were added and expressed as percentage of worms having those specific defects. Or, it can be broken down into defects specific quantification, instead of just number of worms having those specific defects.