Jan 15, 2026
Immunocytochemistry
- Yongjia Duan1
- 1University of California Berkeley
Protocol Citation: Yongjia Duan 2026. Immunocytochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly5j6qvx9/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: January 14, 2026
Last Modified: January 15, 2026
Protocol Integer ID: 238671
Keywords: immunofluorescence, cell cultures, antibodies, fluorescence microscopy, fluorescence microscope, secondary antibody, cultured cell, specific protein
Disclaimer
This protocol was generated by AI
Abstract
This protocol is designed to perform immunofluorescence staining on cultured cells to visualize specific proteins using primary and secondary antibodies, followed by imaging with a fluorescence microscope.
Materials
Glass coverslips (12 mm diameter, sterile) - 24
24-well plates (Sterile, tissue culture treated) - 1
PBS (Phosphate-buffered saline) - 1000 mL
Blocking buffer (1% (w/v) BSA in PBS) - 500 mL
ProLong Gold (Antifade mounting medium) - 1 mL
Reagents:
Fixative Formaldehyde - Crosslinking agent for fixation of cells
Permeabilization Triton X-100 - Detergent for permeabilizing cell membranes
Primary Antibody [Specify Antibody] - Specific antibody for target protein
Secondary Antibody Alexa Fluor - Fluorescently labeled secondary antibody
Nuclear Stain Hoechst 33258 - Stains DNA in cell nuclei
Troubleshooting
Problem
High background fluorescence
Solution
Ensure thorough washing between steps and use appropriate antibody dilutions.
Problem
Weak signal
Solution
Check antibody concentrations and ensure proper incubation times and temperatures.
Safety warnings
Formaldehyde is toxic; handle in a fume hood and dispose of according to regulations. Triton X-100 is an irritant; avoid skin contact. Use gloves and goggles throughout the procedure.
Cell Preparation
Seed cells onto sterile glass coverslips in 24-well dishes.
Washing
Wash coverslips once with 1 mL PBS.
Fixation
Fix cells with 3.7% (w/v) formaldehyde in 200 mM HEPES (pH 7.0) for 10 minutes.
Permeabilization
Wash twice with PBS, then permeabilize cells with 0.1% Triton X-100 in PBS for 5 minutes.
Blocking
Block samples by incubating in blocking buffer (1% BSA in PBS) for 30 minutes at room temperature.
Primary Antibody Incubation
Incubate coverslips with primary antibodies diluted in blocking buffer for 2 hours at 37°C.
Washing
Wash coverslips three times with PBS for 5 minutes each.
Secondary Antibody Incubation
Incubate coverslips with Alexa Fluor-conjugated secondary antibodies in blocking buffer for 1 hour at room temperature.
Nuclear Staining (Optional)
If needed, counterstain nuclei with Hoechst 33258 at a concentration of 1 µg/mL for 5 minutes.
Mounting
Mount coverslips in ProLong Gold and allow to cure.
Imaging
Observations were made with Nikon Eclipse Ti2-E microscope (CFI Plan Apochromat Lambda D 60X Oil objective). Images were processed using Fiji (ImageJ) and Adobe Photoshop Software.
Protocol references