Jun 16, 2026

Immunocytochemistry _ Fluorescent

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4m798lo5/v1
  • 1VanAndelInstitute
Yue Ma
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4m798lo5/v1
Protocol Citation: Yue Ma 2026. Immunocytochemistry _ Fluorescent. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4m798lo5/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: June 16, 2026
Last Modified: June 16, 2026
Protocol  Integer ID: 319289
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Abstract
Fluorescent_cells
Safety warnings
If target protein is a membrane protein, use a less stringent detergent (eg Tween 20 instead of Triton X), or skip permeabilize step.
Immunocytochemistry _ Fluorescent
Wash the cells with PBS for 5 mins, repeat for 3 times.
Fix cells with 10% NBF or 4% PFA for 10min at RT.
Wash cells with PBS for 5 mins, repeat for 3 times. Cells can be stored in PBS at 4°C until staining.
Permeabilize cells with 0.3% Triton X in PBS for 15min.
If target protein is a membrane protein, use a less stringent detergent (eg Tween 20 instead of Triton X), or skip permeabilize step.
Wash cells with PBS for 5 mins, repeat for 3 times.
Blocking: 10% normal goat serum (use a serum that matches the secondary antibody species for best results) + 0.1% Triton X in PBS for 1h at RT.
Primary antibody incubation: dilute antibody in 5% normal goat serum+ 0.1% Triton X in PBS for 24-48h at 4°C.
Wash cells with TBST (0.1% Tween20) for 5 mins, repeat for 5 times.
Secondary antibody incubation: dilute Alexa Fluor 488/546/568/594/633/647 antibody (1:500) in TBST, incubate for 2h at RT or 24h at 4°C.
Wash cells with TBST (0.1% Tween20) for 5 mins, repeat for 3 times.
Counter staining with DAPI or Hoechst 33342 (final concentration 1ug/ml) for 5 min.
Hoechst 33342_MW= 561.93 g/mol, 1ul 20mM stock to 10ml PBS to reach ~1ug/ml.
Rinse the sample several times in PBS.
Drip a drop of antifade reagent on the slide, take the coverslip out of the plate, drain excess buffer from the coverslip and mount on the slide.
Seal the coverslip with transparent nail polish.