Jul 23, 2025
Immunocytochemistry
- Arun Thiruvalluvan1,2,
- Agnete Kirkeby1,2
- 1Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.;
- 2Novo Nordisk Foundation Center for Stem Cell Medicine (renew), University of Copenhagen, 2200 Copenhagen, Denmark
Protocol Citation: Arun Thiruvalluvan, Agnete Kirkeby 2025. Immunocytochemistry . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88m69l2w/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: July 16, 2025
Last Modified: July 23, 2025
Protocol Integer ID: 222628
Keywords: ASAPCRN, immunocytochemistry, fixed cell, cell, protocol
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-024296
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This protocol describes how to perform immunocytochemistry on fixed cells.
Troubleshooting
Fixing
The cells were washed three times with 1xPBS (Thermo Fisher Scientific) and fixed for 15-20 minutes with 4% paraformaldehyde, followed by two washes with 1xPBS. Note: Add 4% (w/v) PFA, enough to cover the cells well (e.g., 100μl to a well in a 96-well plate and 200μl to a well in a 48-well plate) and incubate for 15-20 min. Avoid drying the wells while washing.
Blocking and permeabilization
1h
Non-specific antibody binding sites were blocked using PBS+ (PBS containing 0,1% Triton-X; Thermo Fisher Scientific: HFH10) supplemented with 5% normal goat serum (NGS; Thermo Fisher Scientific: 31872) for 01:00:00 at Room temperature
1h
Staining
2h
Subsequent incubation with primary antibodies diluted in PBS+ with 1%NGS was
performed Overnight at 4 °C
| Antibody target | Provider | Cat# Number | Dilution | |
| LMX1 | Merck Millipore | Cat# AB10533 | 1:1000 | |
| FOXA2 | R&D Systems | Cat# AF2400 | 1:1000 | |
| OTX2 | R&D Systems | Cat# AF1979 | 1:1000 | |
| EN1 | Novo Nordisk | NA | 1:500 | |
| TH | Merck Millipore | Cat# AB152 | 1:1000 | |
| MAP2 | Merck Millipore | Cat# AB5622 | 1:400 | |
| IBA-1 | AbCam | Cat# ab5076 | 1:500 | |
| PU.1 | Cell Signaling | Cat# 2258S | 1:300 | |
| CD45 | Bio Legend | Cat# 304002 | 1:300 |
1h
The next day (Day 2), the primary antibody was removed, and cells were washed three times with 1XPBS. Note: Avoid drying the wells while washing.
Cells were incubated with appropriate Alexa 488 and Cy3-conjugated secondary antibodies and DAPI (1mg/ml; 1:1000) for 01:00:00 at Room temperature , protected from light. Note: Add the 2° antibody-containing solution to the cells (~120 μl/cm²), wrap the plate or chamber slides in aluminium foil to avoid bleaching of the fluorophores, and incubate for 1 h at room temperature (RT) on a shaker. Note: 2° antibody dilution needs to be optimised based on the vendor it's purchased from, we used 1:400 for both Alexa 488 and Cy3-conjugated secondary antibodies.
1h
Wash the cells three times in PBS, leave the stained cells in PBS in the well, and leave the plate wrapped in aluminium foil at 4°C until microscopic analysis. Note: Use PBS containing 0.02% sodium azide (NaN3) to prevent microbial growth. Note: Avoid drying the wells while washing
Cells were washed three times with 1XPBS. Stained cells on chamber slides or mounted on glass slides were imaged using a Leica SP8 confocal laser scanning microscope (CLSM).