Immunofluorescence Staining and High Content Imaging

Wash iPSC derived neurons twice with 1X PBS and fix with 4% PFA for 0h 30m 0s in a 96 well optical bottom plate with a polymer base.

Store fixed neurons at 4C in 1x PBS with 0.02% sodium azide.

Aspirate sodium azide with pipette and permeabilize neurons in 150µL of 1x PBS with 0.5% Triton X-100 for 0h 15m 0s at Room temperature (RT) without shaking.

Aspirate PBST and block neurons with 300µL of 3% BSA and 0.1% Triton X-100 in 1x PBS for 1h 0m 0s at Room temperature.

Aspirate blocking buffer and stain neurons with primary antibodies mouse anti-Synapsin 1 (1:500), goat anti-tdTomato (1ug/ml), and chicken anti-MAP2 (1:5000), in blocking buffer by adding 100µL of blocking buffer for 1h 30m 0sat Room temperature or overnight at 4°C .

Aspirate primary antibody solution and wash samples three times for 0h 5m 0s with 200µL 1x PBS with 0.1% Triton X-100 (0.1% PBST).

Aspirate 0.1% PBST and incubate samples with secondary antibodies in blocking buffer by adding 100µL for 1h 0m 0s at Room temperaturein the dark.

Aspirate secondary antibody solution and wash neurons twice with 200µL of 0.1% PBST for 0h 5m 0s.

Aspirate 0.1% PBST and incubate neurons with DAPI (0.5 mg/ml) by adding 100µL at Room temperature for 0h 10m 0s.

Aspirate DAPI and add 250µL of 1x PBS containing 0.05% sodium azide.

Store plate at 4°C until use.

Allow plate to warm to RT before imaging.

Neurons are imaged using Molecular Devices (San Jose, CA) ImageXpress Micro Confocal High-Content Imaging System at both 20x and 40x.

The laser wavelengths used were DAPI, FITC, Texas Red, and Cy5.

Each well in the 96 well plate was imaged at 8 sites for 40x and 9 sites for 20x with 8-10 z stacks at 1 µm step size. For the 40x objective the pixel size is 0.3438 µm² with a pinhole of 60 µm, 20x objective pixel size is 0.6842 µm² also with a pinhole of 60 µm.

Acquired images are analyzed as 2D maximum projections. Analyze the first two morphometrics, mean number of branches per cell and mean length of neurite outgrowth per cell, with the built-in Neurite Outgrowth Application Module within the MetaXPress 6 software, version 6.7.2.290

Both mean number of neurite branches per cell and the mean length of neurite outgrowth per cell were calculated based on using the DAPI stain as a nuclear marker and the tdTomato stain, which labels excitatory neurons, as the neurite and cell body marker. The values used for subsequent analysis can be optimized depending on staining quality and cell density.

Define cell bodies to have an approximate maximum width of 30 µm, a minimum area 300 µm², and a pixel value of at least 1500 above local background level.

Define nuclei to have an approximate minimum width of 8 µm, an approximate maximum width of 20 µm, and a pixel value of at least 1500 above local background level.

Define neurite outgrowth to have a maximum width of 2 µm, a minimum projection length of 15 µm from the cell body, and a pixel value of at least 500 above local background level.

Acquired images are analyzed as 2D maximum projections. Analyze the third morphometric, excitatory synapse density, using a custom synaptic assay module with MetaXpress 6 software. The values used for subsequent analysis can be optimized depending on staining quality and cell density.

Use Synapsin1 staining to identify puncta with an approximate minimum width of 0.5 µm, an approximate maximum width of 2 µm, and a minimum pixel value of 2500 above local background level.

The custom module will generate the number and area of Synapsin1 positive puncta within the colocalized MAP2+ and tdTomato+ signals.

Puncta density is generated by dividing the total area of puncta within the colocalized MAP2 and tdTomato staining by the area of MAP2+ and tdTomato+ signal within the neurites.