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    Immunocytochemistry

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    meprabhatmishra99
    Immunocytochemistry is a laboratory technique that uses antibodies to detect target proteins in cells. It involves fixing, permeabilizing, and blocking cells, then staining with labeled primary and secondary antibodies. The antibodies are labeled with enzymes or fluorescent dyes to allow visualization of antigen locations in cells. ICC is used for research, diagnosis of diseases like cancer, and differential diagnosis in cytopathology.

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    Immunocytochemistry

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    meprabhatmishra99
    10 views12 pages
    Immunocytochemistry is a laboratory technique that uses antibodies to detect target proteins in cells. It involves fixing, permeabilizing, and blocking cells, then staining with labeled primary and secondary antibodies. The antibodies are labeled with enzymes or fluorescent dyes to allow visualization of antigen locations in cells. ICC is used for research, diagnosis of diseases like cancer, and differential diagnosis in cytopathology.

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    Immunocytochemistry is a laboratory technique that uses antibodies to detect target proteins in cells. It involves fixing, permeabilizing, and blocking cells, then staining with labeled primary and secondary antibodies. The antibodies are labeled with enzymes or fluorescent dyes to allow visualization of antigen locations in cells. ICC is used for research, diagnosis of diseases like cancer, and differential diagnosis in cytopathology.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    10 views12 pages

    Immunocytochemistry

    Uploaded by

    meprabhatmishra99
    Immunocytochemistry is a laboratory technique that uses antibodies to detect target proteins in cells. It involves fixing, permeabilizing, and blocking cells, then staining with labeled primary and secondary antibodies. The antibodies are labeled with enzymes or fluorescent dyes to allow visualization of antigen locations in cells. ICC is used for research, diagnosis of diseases like cancer, and differential diagnosis in cytopathology.

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    of 12

    Immunocytochemistry (ICC)

    Department of Zoology
    University of Lucknow
    Introduction:-
    • Common laboratory assay that can confirm the expression and
    location of target peptides or protein antigens in the cell via
    specific combination of antibodies.
    • Qualitative means of analyzing the subcellular localization of
    target antigens. Chromogenic Detection by
    enzyme-labelled Ab.
    Source- novusbio.com

    • Highly productive method in bio-medical research.


    • This technique began in 1942 by Albert Coons and his co-
    workers. They used fluorescent-labelled antibody to localize
    Pneumococcal antigen in liver sections.
    • Helps to diagnose disease such as cancer.

    Immunofluorescent Detection by
    fluorochrome-labelled Ab.
    Source-novusbio.com
    Immunocytochemistry V/s Immunohistochemistry:-
    As the name suggests,

    • Immunocytochemistry is used to detect antigens in isolated cells,


    where extracellular matrix (ECM) is removed.

    • Whereas, immunohistochemistry is used to detect antigens in


    tissue samples.
    Principle:-
    • ICC uses antibodies to detect the location of
    proteins and other antigens in cell samples.
    • Antibody is linked to a reporter (enzyme,
    fluorophore or fluorescent dye).
    • The antigen–antibody interaction is visualized by
    using either chromogenic detection with a colored
    enzyme substrate, or fluorescent detection with a Source- Bosterbio.com
    fluorescent dye.
    • Reporter  Signal  Detectable by microscope.
    General protocol steps in ICC
    Fixation:-
    • Before fixation, coverslips must be sterilized in autoclave/laminar flow culture
    hood & washed with ethanol.
    • Further, coating matrix (poly-L-lysine) is applied to adhere cells.
    • Fixation of cells  4% formaldehyde/10% Formalin (10 min).
    Why there is need to fix the cells?
    1. Prevent proteolytic enzyme induced autolysis of cells.
    2. Enhance rigidity & mechanical strength of cells.
    3. Preserves cellular morphology & structure.
    • Fixatives – 2 types:
    (a) Cross-linking – Formalin/Formaldehyde (Crosslink proteins via free amine
    groups, forming intermolecular bridges & mask antigens).
    (b) Precipitative – Methanol, acetone, picric acid.
    Permeabilization:-
    • If the epitope of Ag of interest is expressed intracellularly, cellular
    permeabilization necessary for Ab to gain access into the cell.
    • Done by acetone or methanol fixation or by using detergent (Triton X-100).
    • Triton X-100 – Non-ionic, used for permeabilizing the nucleus & mitochondria.
    Blocking:-
    • Ag detection is dependent on specific binding of Ab to its epitope.
    • Binding  Hydrophobic, ionic interactions, H-bonding & other intermolecular
    forces.
    • Unspecific binding sites are blocked by blocking buffer (1 hour at room temp).
    • Best blocking buffer  PBST containing 10% serum from the host of the 2° Ab.
    • 1° Ab is prepared by diluting it in blocking buffer.
    • Incubate at 4°C overnight.
    Immunostaining & mounting:-
    • 1° and 2° Ab conjugated with fluorophore is added into well.
    • Wash off the cells 3 times with PBST.
    • Coverslips are ready to be mounted onto microscope slides & visualized under
    microscope.
    Detection methods:-

    Diagrammatic representation of direct


    and indirect detection
    Applications of ICC:-

    Bio-Medical
    Cytopathology
    Research

    Differential Clinical Application-


    Diagnosis To Detect Cancer

    Urine Cytology- in
    congenital
    infections such as
    congenital HCMV
    References

    • Burry RW. 2010. Immunocytochemistry: a practical guide for biomedical


    research. New York: Springer
    • Immunocytochemistry (ICC) Handbook by Novus Biologicals
    • Maxwell, P. and Salto-Tellez, M. (2016), Validation of immunocytochemistry
    as a morphomolecular technique. Cancer Cytopathology, 124: 540-
    545. https://doi.org/10.1002/cncy.21692

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