Th-NTR-mCherry expression in larvae

(A) Example of a potentially homozygous larvae showing very bright fluorescence under the microscope as compared to (B) weak signals which are indicative of heterozygous lines. Sort the larvae that express very bright red fluorescence.

Embedding larvae in the 96-well plate

A single 5 dpf larvae is embedded per well with 40 μL of low melting point agarose. The zoomed in section shows each larvae is correctly positioned in the center of the well. The red square represents the estimated area that the 20x confocal lens field of view.

Example 96 well plate setup

Each row represents as a single condition (n = 12). The top and bottom row are considered positive (0.2% DMSO), and negative control (MTZ or CBE) with screening compounds + MTZ/CBE in the middle rows.

DA neuron isolation and fluorescence intensity analysis

Example steps in the CellProfiler pipeline that highlights the identification of TH neurons based on the pixel size.

(A) Using the 20x bright field image to overlay the fluorescent image to automatically detect the eyes for removal of autofluorescence.

(B) The “IdentifyPrimaryObjects” command with the global threshold strategy configurations correctly capture the neurons that are later used to quantify intensity.

Bar graph visualization of the screening plate analysis

A t-test between the positive control (blue) and negative control (red) shows significant DA neuron ablation. The difference between the negative control and the different treatment groups (Drug A, B, and C) should be assessed to determine the significance of a hit candidate.

Template for 96-well plate positioning

To assist with the proper alignment and embedding of the dorsal down positioning of the larvae, a custom created template is printed and placed in the bottom of the plate. The red, green, and blue squares represent 20x, 10x and 4x imaging area that the IN Cell 6000 Analyzer can cover. The template is available in the repository (Specific for the Greiner μClear plates).