Dec 25, 2025
Immunofluorescence staining and confocal microscopy in cultured cells
- Xin Xu1,
- Magnus Lam1
- 1Princess Margaret Cancer Centre
Protocol Citation: Xin Xu, Magnus Lam 2025. Immunofluorescence staining and confocal microscopy in cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm177nv3p/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: December 21, 2025
Last Modified: December 25, 2025
Protocol Integer ID: 235612
Keywords: immunofluorescence, confocal microscopy, protein localization, fluorescence imaging, cell biology, proximity ligation assay, PLA, cancer cells, confocal microscopy in cultured cell, transduced cultured cell, cultured cell, cell, cells this protocol, antibody
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes immunofluorescence staining and confocal microscopy in lentivirally transduced cultured cells, including fixation, permeabilization, antibody staining, and mounting for fluorescence imaging.
Guidelines
- Antibody dilutions and incubation times may require optimization depending on antibody performance.
- Avoid drying samples during staining and wash steps.
- PLA experiments should follow the manufacturer’s protocol exactly.
Materials
- Lab-Tek II Chamber 8-well slides (Thermo Fisher Scientific, 154534)
- Alexa Fluor 647-conjugated anti-Rabbit IgG (Thermo Fisher Scientific, A-31573)
- Alexa Fluor 488-conjugated anti-Mouse IgG (Thermo Fisher Scientific, A-11001)
- Triton X-100 (Millipore Sigma, X100-500ML)
- 4% paraformaldehyde (Thermo Fisher Scientific, J61899-AK)
- ProLongTM Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific, P36971)
- glass coverslips (Fisher Scientific, 12541032CA)
Troubleshooting
Before start
- Perform all staining steps at room temperature unless otherwise indicated.
- Prepare all buffers fresh or thaw on the day of use.
- Protect samples from light after secondary antibody incubation.
- Use appropriate biosafety precautions when handling paraformaldehyde.
Cell seeding
Seed lentivirally transduced cells onto Lab-Tek II Chamber 8-well slides (Thermo Fisher Scientific, 154534).
Incubate cells for 24 h to reach 40–60% confluency at the time of fixation.
Fixation
20m
Rinse cells three times with PBS.
Fix cells with 4% paraformaldehyde for 15 min at room temperature.
15m
Wash cells three times with PBS.
5m
Permeabilization
10m
Permeabilize cells with 0.5% Triton X-100 in PBS for 5 min at room temperature.
5m
Wash cells three times with PBS using gentle agitation.
5m
Blocking
1h
Block cells with 3% BSA and 0.1% Tween-20 in PBS for 1 h at room temperature.
1h
Primary antibody incubation
2h
Dilute primary antibodies in 1% BSA and 0.1% Tween-20 in PBS.
Incubate cells with primary antibodies for 2 h at room temperature.
2h
Secondary antibody incubation
5h
Dilute secondary antibodies in 1% BSA and 0.1% Tween-20 in PBS:
- Alexa Fluor 647–conjugated anti-Rabbit IgG (1:1000; Thermo Fisher Scientific, A-31573)
- Alexa Fluor 488–conjugated anti-Mouse IgG (1:1000; Thermo Fisher Scientific, A-11001)
Incubate cells with secondary antibodies for 1.5 h at room temperature, protected from light.
5h
Wash cells three times with PBS.
Mounting
Mount cells with ProLong Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific, P36971).
Cover samples with glass coverslips (Fisher Scientific, 12541032CA).
Cure mounted slides at room temperature, protected from light, for at least 24 h before sealing with nail polish.
Imaging
Acquire images using confocal microscopy under appropriate laser and detector settings.
Proximity ligation assay (PLA) (optional)
Perform the proximity ligation assay (PLA) according to the manufacturer’s instructions (Sigma Aldrich, DUO92101).