Jan 28, 2026
Immunocytochemistry of human iPSC-derived Dopamine Neurons (DaNs)
- Humaira Noor1,2,3,4,
- Kaitlyn Cramb5,2,3,4,
- Richard Wade-Martins5,2,3,4
- 1Nufflied Department of Medicine, Henry Wellcome Building for Molecular Physiology, Old Road, University of Oxford, Oxford OX3 7B, UK;
- 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, UK;
- 3Oxford Parkinson’s Disease Centre, University of Oxford, Oxford OX1 3PT, UK;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 5Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Sherrington Rd, Oxford, OX1 3PT, UK
Protocol Citation: Humaira Noor, Kaitlyn Cramb, Richard Wade-Martins 2026. Immunocytochemistry of human iPSC-derived Dopamine Neurons (DaNs). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn953ql5d/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: February 26, 2025
Last Modified: January 30, 2026
Protocol Integer ID: 123427
Keywords: Dopaminergic neurons, iPSCs, differentiation, immunocytochemistry, derived dopamine neuron, dopamine neuron, synapses in paraformaldehyde, immunocytochemical staining of multiple protein target, immunocytochemistry of human ipsc, synapse, immunocytochemical staining, multiple protein target
Funders Acknowledgements:
Aligning Science Across Parkinson’s Collaborative Research Network
Grant ID: ASAP-020370, ASAP-025192
Abstract
This protocol outlines the immunocytochemical staining of multiple protein targets for characterisation and synapses in Paraformaldehyde (PFA)-fixed cells plated in a 96-well format plate.
Materials
Reagents:
- Phosphate-buffered saline, pH 7.4 (PBS) (Life Technologies, CAT# 10010056)
- Triton X-100 (Sigma- Aldrich, SKU# X100)
- Paraformaldehyde (PFA) (Sigma- Aldrich, CAT#P6148)
- DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) (Thermo Fisher Scientific, CAT# D1306)
Primary Antibodies:
- Rabbit anti-Foxa2 (Abcam, CAT# ab108422)
- Rabbit anti-Homer1 (Synaptic Systems, CAT# 160 003)
- Guinea Pig anti-synapsin I/II (Synaptic Systems, CAT# 106 004)
- Chicken anti-Map2 (Abcam, CAT# ab92434)
- Sheep anti-TH (Sigma-Aldrich, CAT#ab1542)
- Rabbit anti-vGLUT2 (Synaptic Systems, CAT# 135 403)
- Rabbit anti-vMAT2 (Thermo Fisher Scientific, CAT# PA5-22864)
Secondary Antibodies:
- Donkey anti-mouse IgG Alexa Fluor 488 (Molecular Probes, CAT# A-21202)
- Donkey anti-chicken IgG Alexa Fluor 555 (Thermo Fisher Scientific, CAT# A78949)
- Donkey anti-rabbit IgG Alexa Fluor 647 (Molecular Probes, CAT# A-31573)
- Goat anti-guinea pig IgG Alexa Fluor 488 (Abcam, CAT# ab96954)
- Goat anti-chicken IgG Alexa Fluor 555 (Thermo Fisher Scientific, CAT# A32932)
- Goat anti-rabbit IgG Alexa Fluor 647 (Molecular Probes, CAT# A-21245)
Equipment:
- 96 well tissue culture treated plate (Greiner Bio-One Ltd, CAT# 655090)
- Opera Phenix™ Plus high-content imaging system (Revvity; CAT# HH14001000) - https://www.revvity.com/gb-en/product/opera-phenix-plus-system-hh14001000
SOLUTIONS
Preparing f 4% PFA fixing solution:
Mix 10 mL of 16% PFA with 30 mL PBS.
Preparing 10% Normal Serum:
Add 1 mL of Normal Serum (NS) in PBS.
**Donkey Serum for characterisation staining and Goat serum for synaptic staining**
Preparing 0.01% Triton X-100 solution:
Add 2 µL of Triton X-100 solution in 20 mL 10% Normal Serum (NS).
Troubleshooting
Safety warnings
Cells must be fixed with PFA under a fume hood. Any materials (e.g., pipette tips, reservoirs, or waste containers) that come in contact with PFA should remain inside the fume hood for a minimum of 24 hours before discarding. PFA waste should be discarded as per local guidelines.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
Cells should be plated at a density of 50 000 cells/well in a 96 area full area plate which has been precoated with Geltrex.
Additionally, care should be taken when adding and removing liquid as these adherent cells are prone to peeling. If the plates are stored prior to staining, a plastic strip should be used to seal the wells to prevent evaporation or contamination, and the plates should be kept at 4°C.
PFA Fixation
Remove the media from the wells to be fixed.
Add 50ul of 4% PFA solution to the wells and keep it for 5 minutes for synaptic markers and incubate for 5 minutes for synaptic markers or 10 minutes for other markers at room temperature (RT).
Remove PFA and wash the cells twice with PBS.
Permeabilisation and Blocking
Permeabilise the cells with 0.01% Triton X-100 for 1 hour.
Remove the solution and wash with PBS.
Primary Antibody Incubation
Prepare the primary antibody solution in 1% NS in PBS, supplemented with the appropriate primary antibodies (see Materials) at their respective working concentrations as in Table 1.
| A | B | |
| Primary Antibodies | Dilution | |
| Rabbit anti-Foxa2 | 1:500 | |
| Rabbit anti-Homer I | 1:500 | |
| Guinea Pig anti-Synapsin I/II | 1:500 | |
| Chicken anti-Map2 | 1:500 | |
| Sheep anti-TH | 1:500 | |
| Rabbit anti-vGLUT2 | 1:500 | |
| Rabbit anti-vMAT2 | 1:500 |
Table 1 - Dilution factors for Primary Antibodies used.
Add 50 µl of primary antibody solution to each well and incubate overnight at 4°C.
Secondary Antibody Incubation
Remove the primary antibody solution and wash twice with PBS.
Incubate in species-appropriate Alexa Fluor® secondary antibodies as mentioned in Table 2 with DAPI (1:10000) in 1% NS in PBS for 1 hour at room temperature.
| A | B | |
| Secondary Antibodies | Dilution | |
| Donkey anti-mouse IgG Alexa Fluor 488 | 1:1000 | |
| Donkey anti-chicken IgG Alexa Fluor 555 | 1:1000 | |
| Donkey anti-rabbit IgG Alexa Fluor 647 | 1:1000 | |
| Goat anti-guinea pig IgG Alexa Fluor 488 | 1:1000 | |
| Goat anti-chicken IgG Alexa Fluor 555 | 1:1000 | |
| Goat anti-rabbit IgG Alexa Fluor 647 | 1:1000 |
Table 2 - Dilution factors for Secondary Antibodies used.
Image Acquisition
Acquire wells images using a 40x magnification water immersion objective Opera Phenix High content screening system, capturing images at a focus level of +1 µm and a binning factor of 1 for synaptic imaging.
For characterisation, acquire images using 40x magnification water immersion objective, capturing images at five focal planes ranging from +1 µm to +9 µm and a binning factor of 2.
Take 20 images on average per well, with 3-5 wells analysed per cell line per differentiation.