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HOME > Learn > Principle and method of the experiment > The principle and method of Immunocytochemistry (IC)

IHCとは
IC, or Immunocytochemistry, is a method used to detect antigens within cells using antibodies. By utilizing the specificity of antibodies, antigens can be detected and their intracellular localization can be observed under a microscope. Fluorescence is often used for detection in IC. Here is one protocol that can be used for fluorescent cell staining.



Types of Fluorescent Labels

　■ Main fluorescence substances
Fluorescence substances
Fluorescent dyes
(small molecular dyes)		 FITC (Fluorescein Isothiocyanate), Alexa Fluor® dyes, Cy dyes, etc.
Fluorescent proteins PE (Phycoerythrin), APC (Allophycocyanin), etc.





Direct Method vs. Indirect Method

There are two methods for detecting labeled antibodies: directly labeling the primary antibody with a fluorescent label (direct method), or using a fluorescently labeled secondary antibody that reacts with the primary antibody (indirect method). In direct method, multiple staining is easily achievable, but combinations of fluorescent dyes that do not interfere with each other needs to be considered.

The principle and method





Advantages Disadvantages
Direct Method
  • • Easy for multiple staining, especially when using fluorescently labeled primary antibodies.
  • • Non-specific staining from secondary antibodies can be prevented.
  • • Shorter working time.
    		•
  • • Higher cost due to the requirement of using as many labeled antibodies as the target molecules.
  • • Availability of labeled antibodies for the desired targets may be limited.
  • • Some antibodies may lose activity due to labeling.
    		•
    Indirect Method
  • • Higher versatility as the same labeled secondary antibodies can be used with primary antibodies from the same host species.
    		•
  • • For multiple staining, it is necessary for the primary antibodies to come from different host animals, which can make it difficult to detect multiple target molecules simultaneously compared to the direct method.
  • • Longer reaction time required for secondary antibodies compared to the direct method.
  • • Non-specific staining may occur due to secondary antibodies.
    		•


    Procedure for Fluorescent Immunostaining (Indirect Method)

    ※An example performed at MBL

    ↓ Cell seeding

    ↓ Cell fixation (using 4% paraformaldehyde, formalin, etc.)

    ↓ Washing

    ↓ Permeabilization (e.g. using surfactants)

    ↓ Washing

    ↓ Blocking

    ↓ Primary antibody reaction (at room temperature for 1-2 hours)

    ↓ Washing

    ↓ Fluorescent or enzyme-labeled secondary antibody reaction (at room temperature for 1 hour)

    ↓ Washing

    ↓ Counterstaining (e.g. with DAPI)

    ↓ Observation under a fluorescence microscope


    Meaning of each step

    ▌ Cell fixation
    To stabilize cell morphology and inactivate enzymes. The optimal fixation method depends on the antigen or antibody used, so the type of fixative, fixation time, and temperature should be considered for the best results.

    　■ Types of Fixatives
    Aldehyde-based Formaldehyde, paraformaldehyde, and formalin (aqueous solution of formaldehyde) are the most commonly used cross-linking agents. They react with the amino group (-NH2) of proteins and nucleic acids to form reversible bonds. Paraformaldehyde is sometimes prepared and used as a fixative.
    Organic solvents Organic solvents such as methanol, ethanol, and acetone can also be used as fixatives. In this case, lipids inside the cells are removed, so they cannot be used for detecting lipid-bound proteins such as membrane proteins.

    ▌ Membrane permeabilization
    To allow antibodies to penetrate the cell membrane and nuclear membrane after fixation. Surfactants like Triton X-100 or NP-40 are commonly used, and the concentration and duration of treatment should be adjusted based on the target protein.

    ▌ Blocking
    To prevent non-specific staining by primary and secondary antibodies. Blocking agents like BSA or serum from the same host animal as the secondary antibody are used.

    ▌ Counterstaining
    To visualize specific cellular structures by staining them with a color contrasting to the target protein. DAPI or Hoechst stains are commonly used to visualize the shape of the nucleus.


    Items needed for experiment

    • Cells to be stained (preferably adherent cells)
    • Glass bottom dishes
    • Substances to enhance cell adhesion (such as poly-L-lysine, collagen)
    • Cell fixation solution (4% paraformaldehyde, formalin, methanol, etc.)
    • Surfactants (NP-40, Triton X-100, Tween20, etc.)
    • Blocking agents (BSA, serum, etc.)
    • PBS
    • 0.05-0.2% Tween20/PBS (PBS-T)
    • Primary antibodies
    • Fluorescently labeled secondary antibodies
    • DAPI
    • Water-soluble mounting medium


    Method for Cell Culturing and Sample Preparation

    ※An in-house example of preparing cell samples using NRK cells will be introduced.

    1. Cell culturing

    cellcountCultivate NRK cells in a 10 cm dish. When cells reach approximately 70% confluency, collect and count the cells.




    2. Cell seeding - (1)

    Place an 18 mm x 18 mm coverslip autoclaved on a 6-well culture slide.




    3. Cell seeding - (2)

    Add 200 µL of the cell suspension adjusted to 5 x 105 cells/mL onto the coverslip（1 x 105/well）.
    Incubate for about 1 hour at 37°C in a 5%CO2 incubator.



    4. Cell seeding - (3)

    Add 2 mL of medium to each well and further incubate overnight.




    5. Cell seeding - (4)

    cellcount※If there is no need for specific treatment other than autophagy comparison, proceed to 6.cell fixation.

    In the following day, remove the medium by aspiration after confirming cell adhesion under a microscope. Add 200 µL of 10% FCS-RPMI to the nutrient well and 200 µL of RPMI to the starved well. Incubate for about 3 hours at 37°C in a 5%CO2 incubator.


    6. Cell fixation

    Confirm cell adhesion under a microscope. Discard the medium, wash the cells with PBS, add 200 µL of 4% paraformaldehyde solution slowly, and let it stand at room temperature for 10 minutes.



    7. Membrane permeabilization

    Wash the cells twice with PBS, add 200 µL of 100 µg/mL digitonin in PBS to each well, and let it stand at room temperature for 10 minutes.


    8. Primary antibody reaction

    Wash the cells twice with PBS after removing the supernatant, add the primary antibodies diluted in PBS (200 µL each), and let it react at room temperature for 1 hour.




    9. Fluorescent or enzyme-labeled secondary antibody reaction

    Wash the cells three times with PBS, add the secondary antibodies diluted 500 times in PBS (200 µL each), cover with aluminum foil to block light, and let it react at room temperature for 30 minutes.



    10. Counterstaining

    Wash the cells three times with PBS, add DAPI (1 µg/mL, 100 µL), block light, and let it react at room temperature for 30 minutes.




    11. Mounting

    Wash the cells three times with PBS, mount them, and observe under a fluorescence microscope.












    Related links

    Fractionation and purification of proteins


    Immunostaining