TITLE: Immunofluorescence Staining and High Content Imaging
AUTHORS: David Sirkin, Gregory Tracy, Hanwen Zhang, Lilia Peyton, Ada McCarroll, Jubao Duan

[DESCRIPTION]
This protocol offers a description of immunofluorescence staining and high content imaging of iPSC induced neurons using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, San Jose, CA).

[STEPS]
SECTION: Immunofluorescence Staining
1. Wash iPSC derived neurons twice with 1X PBS and fix with 4% PFA for 30 min in a 96 well optical bottom plate with a polymer base.

SECTION: Immunofluorescence Staining
2. Store fixed neurons at 4C in 1x PBS with 0.02% sodium azide.
SECTION: Immunofluorescence Staining
3. Aspirate sodium azide with pipette and permeabilize neurons in 150 µL of 1x PBS with 0.5% Triton X-100 for 15 min at Room temperature (RT) without shaking.

SECTION: Immunofluorescence Staining
4. Aspirate PBST and block neurons with 300 µL of 3% BSA and 0.1% Triton X-100 in 1x PBS for 60 min at Room temperature.

SECTION: Immunofluorescence Staining
5. 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 100 µL of blocking buffer for 90 minat Room temperature or overnight at  4 °C .

SECTION: Immunofluorescence Staining
6. Aspirate primary antibody solution and wash samples three times for 5 min with 200 µL 1x PBS with 0.1% Triton X-100 (0.1% PBST).

SECTION: Immunofluorescence Staining
7. Aspirate 0.1% PBST and incubate samples with secondary antibodies in blocking buffer by adding 100 µL for 60 min at Room temperaturein the dark.

SECTION: Immunofluorescence Staining
8. Aspirate secondary antibody solution and wash neurons twice with 200 µL  of 0.1% PBST for 5 min.

SECTION: Immunofluorescence Staining
9. Aspirate 0.1% PBST and incubate neurons with DAPI (0.5 mg/ml) by adding 100 µL  at Room temperature for 10 min.

SECTION: Immunofluorescence Staining
10. Aspirate DAPI and add 250 µL of 1x PBS containing 0.05% sodium azide. 

SECTION: Immunofluorescence Staining
11. Store plate at 4 °C until use.

SECTION: Image Acquisition
12. Allow plate to warm to RT before imaging.
SECTION: Image Acquisition
13. Neurons are imaged using Molecular Devices (San Jose, CA) ImageXpress Micro Confocal High-Content Imaging System at both 20x and 40x.
SECTION: Image Acquisition
14. The laser wavelengths used were DAPI, FITC, Texas Red, and Cy5.
SECTION: Image Acquisition
15. 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.

SECTION: 20x Image Analysis
16. 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
SECTION: 20x Image Analysis
17. 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.
SECTION: 20x Image Analysis
17.1. 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. 
SECTION: 20x Image Analysis
17.2. 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. 
SECTION: 20x Image Analysis
17.3. 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.
SECTION: 40x Image Analysis
18. 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.
SECTION: 40x Image Analysis
18.1. 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.
SECTION: 40x Image Analysis
19. The custom module will generate the number and area of Synapsin1 positive puncta within the colocalized MAP2+ and tdTomato+ signals.
SECTION: 40x Image Analysis
19.1. 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.