Immunocytochemistry
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We have listed the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.
Abstract
Immunocytochemistry is a technique that uses antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with a primary antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the steps for fixing and labeling HeLa cells for a target protein using the formaldehyde fixation method. The protocol may need to be optimized for different cells, target proteins, etc.
Before start
See the Materials section for preparation of necessary stock solutions.
Refer to the manufacturer's instructions for additional information specific to your antibodies, such as antibody concentrations, incubation times, and recommended compatible reagents.
Secondary antibodies must match the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary antibodies raised in a mouse.
Steps
Seeding cells
Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated plate.
Seed 5*10^3 HeLa cells per well.
Allow the HeLa cells to grow to the desired density before labeling.
Fixing and permeabilizing cells
Gently aspirate the media from the 24-well plate.
Wash each well with 500µL of PBS, remove the wash, and dispose of it in an appropriate waste container.
Fix each well with 500µL of cold 4% paraformaldehyde in PBS on ice for 0h 15m 0s.
Remove the paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container.
Wash 3x for 0h 5m 0s in 500µL PBS on a rocking platform.
Permeabilize cells for 0h 10m 0s at Room temperature on a rocking platform in 500µL permeabilization buffer.
Remove the permeabilization buffer and dispose of it in an appropriate waste container.
Wash 3x for 0h 5m 0s in 500µL PBS on a rocking platform.
Labeling with antibody
Block for 0h 20m 0s at Room temperature on a rocking platform in 500µL blocking buffer.
Remove the blocking buffer and dispose of it in an appropriate waste container.
Dilute the primary antibody to the desired concentration in antibody dilution buffer.
Add 500µL of the diluted antibody to the wells and incubate 2h 0m 0s at Room temperature.
Remove the primary antibody and dispose of it in an appropriate waste container.
Wash 3x for 0h 5m 0s in 500µL PBS on a rocking platform.
Dilute the fluorescently-labeled secondary antibody to the desired concentration in antibody dilution buffer.
Add 500µL fluorescently-labeled secondary antibody to the wells and incubate 0h 30m 0s at Room temperature in the dark.
Remove the secondary antibody and dispose of it in an appropriate waste container.
Wash 3x for 0h 5m 0s in 500µL PBS on a rocking platform.
(Optional) Counterstain nuclei with 500µL of 300 nM DAPI working solution for 0h 10m 0s at Room temperature in the dark.
Remove the DAPI and dispose of it in an appropriate waste container.
Wash 3x for 0h 5m 0s in 500µL PBS on a rocking platform.
Use tweezers to gently remove the coverslip.
Blot the coverslip with a laboratory wipe to remove excess liquid.
Add 1 drop of anti-fade mounting medium to the microscope slide.
Gently place the coverslip on the microscope slide with the cell side facing down.
Observe the cell labeling on a microscope with appropriate fluorescent filters.
