TITLE: Immunocytochemistry
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
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.

[GUIDELINES]
Workflow Timeline:
Day 1: Seed cells
Day 3-4: Fix and label cells

Tips and Troubleshooting:
We recommend wiping down all pipettes and equipment with 10% bleach prior to use. 

The optimal fixation method will vary depending on the sample type and the protein of interest. You may need to try a variety of fixation methods to find the best conditions for your target.

The optimal antibody concentration will vary between antibodies. Review the manufacturer's instructions before starting your experiment and consider titrating your antibody to determine the optimal  dose.

To ensure that your antibody is both functioning as expected and specific, include a positive control sample that you know expresses the protein, such as cells transfected with a plasmid to express the  protein of interest, and a negative control sample such as cells that do not express the protein of interest.

[STEPS]
SECTION: Seeding cells
1. Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated plate.
SECTION: Seeding cells
2. Seed 5*10^3 HeLa cells per well.
SECTION: Seeding cells
3. Allow the HeLa cells to grow to the desired density before labeling.
SECTION: Fixing and permeabilizing cells
4. Gently aspirate the media from the 24-well plate.
SECTION: Fixing and permeabilizing cells
5. Wash each well with 500 µL of PBS, remove the wash, and dispose of it in an appropriate waste container.

SECTION: Fixing and permeabilizing cells
6. Fix each well with 500 µL of cold 4% paraformaldehyde in PBS on ice for 15 min.
 



SECTION: Fixing and permeabilizing cells
7. Remove the paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container.
SECTION: Fixing and permeabilizing cells
8. Wash 3x for 5 min in 500 µL PBS on a rocking platform.

SECTION: Fixing and permeabilizing cells
9. Permeabilize cells for 10 min at Room temperature on a rocking platform in 500 µL permeabilization buffer.

SECTION: Fixing and permeabilizing cells
10. Remove the permeabilization buffer and dispose of it in an appropriate waste container.
SECTION: Fixing and permeabilizing cells
11. Wash 3x for 5 min in 500 µL PBS on a rocking platform.

SECTION: Labeling with antibody
12. Block for 20 min at Room temperature on a rocking platform in 500 µL blocking buffer.

SECTION: Labeling with antibody
13. Remove the blocking buffer and dispose of it in an appropriate waste container.
SECTION: Labeling with antibody
14. Dilute the primary antibody to the desired concentration in antibody dilution buffer.
 

SECTION: Labeling with antibody
15. Add 500 µL of the diluted antibody to the wells and incubate 120 min at Room temperature.

SECTION: Labeling with antibody
16. Remove the primary antibody and dispose of it in an appropriate waste container.
SECTION: Labeling with antibody
17. Wash 3x for 5 min in 500 µL PBS on a rocking platform.

SECTION: Labeling with antibody
18. Dilute the fluorescently-labeled secondary antibody to the desired concentration in antibody dilution buffer.
 

SECTION: Labeling with antibody
19. Add 500 µL fluorescently-labeled secondary antibody to the wells and incubate 30 min at Room temperature in the dark.
 




SECTION: Labeling with antibody
20. Remove the secondary antibody and dispose of it in an appropriate waste container.
SECTION: Labeling with antibody
21. Wash 3x for 5 min in 500 µL PBS on a rocking platform.

SECTION: Labeling with antibody
22. (Optional) Counterstain nuclei with 500 µL of 300 nM DAPI working solution for 10 min at Room temperature in the dark.

SECTION: Labeling with antibody
23. Remove the DAPI and dispose of it in an appropriate waste container.
SECTION: Labeling with antibody
24. Wash 3x for 5 min in 500 µL PBS on a rocking platform.

SECTION: Labeling with antibody
25. Use tweezers to gently remove the coverslip.
SECTION: Labeling with antibody
26. Blot the coverslip with a laboratory wipe to remove excess liquid.
SECTION: Labeling with antibody
27. Add 1 drop of anti-fade mounting medium to the microscope slide.

SECTION: Labeling with antibody
28. Gently place the coverslip on the microscope slide with the cell side facing down.
SECTION: Labeling with antibody
29. Observe the cell labeling on a microscope with appropriate fluorescent filters.