Immunocytochemistry (ICC) protocol

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Immunocytochemistry (ICC) protocol

1. Cell Preparation:
   • Prepare and culture the cells of interest on appropriate culture dishes or coverslips.
   • Ensure that the cells reach the desired confluence and adhere properly to the substrate.
2. Fixation:
   • Remove the growth medium and gently wash the cells with phosphate-buffered saline (PBS) to remove any residual media or serum.
   • Fix the cells by adding a suitable fixative (e.g., 4% paraformaldehyde) to cover the cells completely.
   • Incubate the cells in fixative for a specific duration (typically 10-20 minutes) at room temperature or as recommended for your specific fixative.
3. Permeabilization:
   • After fixation, wash the cells three times with PBS to remove the fixative.
   • Treat the cells with a permeabilization agent such as 0.1-0.3% Triton X-100 in PBS for 5-10 minutes to allow antibody penetration.
4. Blocking:
   • Block non-specific binding sites by incubating the cells with a blocking solution (e.g., 5% bovine serum albumin or 10% normal serum in PBS) for 1 hour at room temperature.
   • This step helps reduce background staining.
5. Primary Antibody Incubation:
   • Dilute the primary antibody (specific to your target antigen) in a suitable diluent (e.g., blocking solution or antibody dilution buffer).
   • Add the diluted primary antibody solution to the cells, ensuring complete coverage.
   • Incubate the cells with the primary antibody at the recommended temperature and duration (usually overnight at 4°C or 1-2 hours at room temperature).
6. Washing:
   • After primary antibody incubation, wash the cells thoroughly with PBS to remove unbound antibodies.
   • Repeat the washing step at least three times, with each wash lasting for 5 minutes.
7. Secondary Antibody Incubation:
   • Prepare the secondary antibody by diluting it in the appropriate diluent.
   • Add the diluted secondary antibody solution to the cells, ensuring complete coverage.
   • Incubate the cells with the secondary antibody for the recommended time at room temperature in the dark.
8. Washing:
   • Wash the cells again with PBS to remove unbound secondary antibodies.
   • Repeat the washing step three times, each time for 5 minutes.
9. Optional: Nuclear Staining:
   • If desired, stain the cell nuclei with a DNA-specific dye, such as DAPI or Hoechst, following the manufacturer’s instructions.
   • Incubate the cells with the nuclear stain for the recommended time.
10. Mounting:

• Carefully remove excess liquid from the cells.
• Apply an appropriate mounting medium to cover the cells and prevent sample drying.
• Place a coverslip over the mounting medium and seal the edges with nail polish or a commercial sealant.

11. Imaging:

• Allow the mounting medium to dry and harden (if applicable).
• Image the stained cells using a fluorescence microscope or other appropriate imaging system.
• Adjust the microscope settings, such as excitation wavelengths and filters, to visualize the fluorophores used.

Note: This protocol provides a general outline, and specific steps may vary depending on the experimental requirements, cell type, and target antigens. It’s essential to refer to the antibody manufacturer’s instructions and optimize the protocol based on your specific experimental conditions.