Apr 06, 2024
Immunofluorescence Staining and High Content Imaging
- David Sirkin1,
- Gregory Tracy1,
- Hanwen Zhang1,
- Lilia Peyton1,
- Ada McCarroll1,
- Jubao Duan2
- 1Endeavor Health/NorthShore University HealthSystem;
- 2NorthShore University HealthSystem/University of Chicago
Protocol Citation: David Sirkin, Gregory Tracy, Hanwen Zhang, Lilia Peyton, Ada McCarroll, Jubao Duan 2024. Immunofluorescence Staining and High Content Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v928o4l3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2024
Last Modified: April 06, 2024
Protocol Integer ID: 97722
Abstract
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).
Materials
96 well optical bottom plate with a polymer base (Fisher Scientific, 12-566-70).
1x PBS, pH 7.4 (Fisher Scientific, 10-010-023)
Sodium Azide 5% (Avantor, BDH7465-2)
Triton X-100 (Sigma, SLCD3244)
Bovine Albumin Fraction V (7.5% solution) (Fisher Scientific, 50-121-5315)
DAPI (1mg/ml) (Fisher Scientific, EN62248)
Primary Antibodies
| Antibody | Catalogue Number | Concentration | |
| Mouse anti-Synapsin 1 | SySy, 106011 | 1:1,000 | |
| Goat anti-tdTomato | Thermofisher, TA150129 | 1ug/ml | |
| Chicken anti-MAP2 | Sigma, AB5543 | 1:5,000 |
Secondary Antibodies
| Antibody | Catalogue Number | Concentration | |
| Donkey anti mouse Alexa 488 | Fisher Scientific, A211202 | 1:1,000 | |
| Donkey anti-chicken Alexa 647 | Fisher Scientific, EN62248 | 1:1,000 | |
| Donkey anti-goat Alexa 568 | Fisher Scientific, A11057 | 1:1,000 |
Immunofluorescence Staining
Immunofluorescence Staining
Wash iPSC derived neurons twice with 1X PBS and fix with 4% PFA for 00:30:00 in a 96 well optical bottom plate with a polymer base.
30m
Store fixed neurons at 4C in 1x PBS with 0.02% sodium azide.
Aspirate sodium azide with pipette and permeabilize neurons in 150 µL of 1x PBS with 0.5% Triton X-100 for 00:15:00 at Room temperature (RT) without shaking.
15m
Aspirate PBST and block neurons with 300 µL of 3% BSA and 0.1% Triton X-100 in 1x PBS for 01:00:00 at Room temperature .
1h
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 01:30:00 at Room temperature or overnight at 4 °C .
1h 30m
Aspirate primary antibody solution and wash samples three times for 00:05:00 with 200 µL 1x PBS with 0.1% Triton X-100 (0.1% PBST).
5m
Aspirate 0.1% PBST and incubate samples with secondary antibodies in blocking buffer by adding 100 µL for 01:00:00 at Room temperature in the dark.
1h
Aspirate secondary antibody solution and wash neurons twice with 200 µL of 0.1% PBST for 00:05:00 .
5m
Aspirate 0.1% PBST and incubate neurons with DAPI (0.5 mg/ml) by adding 100 µL at Room temperature for 00:10:00 .
10m
Aspirate DAPI and add 250 µL of 1x PBS containing 0.05% sodium azide.
Store plate at 4 °C until use.
Image Acquisition
Image Acquisition
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.
20x Image Analysis
20x Image Analysis
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.
40x Image Analysis
40x Image Analysis
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.