Immunohistochemistry,

Immunocytochemistry

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Definition

Immunohistochemistry:

The method aiming to detect tissue antigens (proteins, glycoproteins and lipoproteins) with specific in situ immundetection. (In broader aspect the in situdetection of DIG and biotinylated probes with specific antibody is also belong to this category).

Immunocytochemistry:

Detection of surface antigens (markers) on isolated cells (ie. membrane proteins on blood cells).

The detection is based on specific antigen-antibody binding (immunreactions). Aspecific antibody that was produced by single B cell clone identifies an epitopewith 8-15 length aminoacid sequence in a protein.

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Main application fields: - Cell and tissue typing (ie. for transplantation).

- Cell identification and isolation.

- In Pathology the immunohistochemical reactions are widely applied:

- Tumor diagnostic: tissue or cell origin identification of a tumor, determination of differentiation stage.

- Presence detection of pathogens (i.e.. detection bacteria, viruses or their antigens).

- Specific detection of receptors (i.e. the strategy of treatment of breast cancer depends on the expression estrogens or progesterone receptor types, or it expresses the HER-2/neu antigen etc.)

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Cell (tissue) determinants - markers

The immunohistochemical detections are most frequently target the following type of markers:

– Skin markers, cytokeratines (CK)

– Connective tissue markers

– Hormones, hormone receptors

– Melanoma markers

– Lymphoma markers

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Main methodical steps of immunohistochemistry

- Tissue or (cell) fixation

- Antigen unmasking

- Blocking

- Selection of appropriate detection signals

- Parallel detection of more antigens

- Background staining

- Controls

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Labeling method in immunocytochemistry

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Carcinoembryonic antigen (CEA): used for identification of adenocarcinomas.

Cytokeratins: used for identification of carcinomas but may also be expressed in some sarcomas.

CD15 and CD30 : used for Hodgkin's disease

Alpha fetoprotein: for yolk sac tumors and hepatocellular carcinoma

CD117 (KIT): for gastrointestinal stromal tumors (GIST)

Prostate specific antigen (PSA): for prostate cancer

Estrogens and progesterone staining for tumour identification

Identification of B-cell lymphomas using CD20Identification of T-cell lymphomas using CD3

Some Diagnostic IHC markers

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Tissue-, cell sample fixation

Aim: To attach (or immobilize) the tissue specimens, molecules to a solid surface, to block the autolysis processes, or harden the tissue block for successful slicing.

Types of fixation materials:

crosslinkers(formaldehyde, glutaraldehyde)

coagulating fixation(acetone, methanol)

or the mixture of above listed materials.

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Slice preparation from tissue

Elaboration processes of fixed organ

or tissue samples.

- decalcification

- slicing from frozen specimens

- embedding into paraffin and

slicing

scrapes, prints and smears can also be made.

In the procedure, depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) penetrable slices are taken of the tissue of interest. The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides.

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Antigen retrieval(unmasking)

Antigen retrieval: Antigen retrieval (or antigen recovery) is performed to expose or retrieve antigen epitopes which have become masked by the tissue fixation process without modifying the fine structure of the tissue.

During formaldehide tisssue fixation the natural conformation of the protein are changed, irreversible crosslinks are established, causing the possibility that the antigen determinants (epitopes) cannot be accessed by antibody molecules.

The epitope is a tiny molecular structure (frequently smaller that the determinant) of antigen molecule which will start a specific reaction with the antibody. The paratoppossesses a similar structure with the epitope, therefore might be the source of crossreaction.

The protocol of antigen unmasking must be standardized (pH, temperature, retrieval time, heating method). A microwave oven or pressure cooker are frequently used for unmasking.

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Blocking (inhibition of non specific binding)

Likewise to other immuntests (Western-blot, ELISA) in immunohystochemical studies it is also important to avoid the non-specific binding of the antibodies (antigen independent binding).

Before adding the antibodies to tissue slices, the specimens must be preincubated with a blocking protein (BSA, milk protein). Very often the blocking protein is also added to the solution which contain the primary antibody.

The efficiency of blocking should be checked with parallel control samples from which the primary (antigen specific) antibody is omitted.

Preincubation of the primary antibody with control peptide (it was used for the antibody induction) is a possibility to confirm the specific binding.

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Applied antibody typesThe antibodies used for specific detection can be polyclonal or monoclonal:

Monoclonal antibodies are generally considered to exhibit greater specificity. (crossreactions can be avoided).

Polyclonal antibodies are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes.

Antibodies can also be classified as primary or secondary reagents:

Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against primary antibodies.

The secondary antibodies recognize immunoglobulins of a particular species and are conjugated to either biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase (HRP). Some secondary antibodies are conjugated to fluorescent agents, such as the Alexa Fluor or Dylight Fluor family, are also frequently used for detection of proteins in IHC procedures.

Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest.

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Direkt IHCThere are two strategies used for the immunohistochemical detection of antigens in tissue:

In both cases, the tissue is treated to rupture the membranes, usually by using a kind of detergent such as Triton X-100. Some antigens also need an additional step for unmasking, resulting in better detection results.

The direct method is a one-step staining method, and involves a labeled antibody (e.g. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is therefore simple and rapid. However, it can suffer problems with sensitivity due to little signal amplification, therefore less commonly used than the indirect method.

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Indirekt IHCThe indirect method involves two antibodies:

an unlabeled primary antibody which reacts with tissue antigen,

and a labeled secondary antibody which reacts with the primary antibody. (The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised.)

Advantage:

1. This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second antibody can be labeled with a fluorescent dye or an enzyme.

2. By applying control samples from which the primary antibody is omitted, the non specificbinding capability of the secondary antibodyto the tissue sample can be checked.

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Frequent enzyme-substrate systems

Peroxidase (HRPO):

Diaminobenzidin (DAB) – brown

Aminoethyl-carbasol (AEC)- red

True Blue – blue

Alkaline phosphatase (ALP):

Nitroblue tetrasolium (NBT) – blue

Bromo-chloro-indoyl phosphate (BCIP) - blue

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Streptavidin/peroxidase detection system

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Color scale of chromogen substrates

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Staining examples

Lymphatic node

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The expression of Claudin (tight junction) proteins in gingiva

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The evaluation of immunhistochemistry

Three main aspects:

1. Specificity

2. Sensitivity

3. Reproducibility

It is important: to distinguishthe real reaction products andthe pseudo-positive, or the pseudo-negative artifacts

Visualization of an apoptotic cell

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- Fluorescens microscope

- Laser Scanning Confocal microscope

Equipments applied for the detection of the fluorescent labeling

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Sample images made by confocal microscope

HeLa Cells Rat cerebellum

Cardiomyocyte

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Frog astrocyte

Mitotic spindle

Sample images made by confocal microscope

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Cardiac myocytes labeled with rhodamine-phalloidin which labels F-actin filaments

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3D modelling

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Protein collocalization

Osteosarcoma cells were staning with fluorescein labeled anti-tubulin antibody (green) and F-actin filaments were stained with rhodamine-phalloidin.

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Application of immunocytochemistry for cell groups identification and sorting

- FluorocytometryIdentification of cell groups

based their surface markers (receptors)

- Fluorescence-Activated Cell Sorting (FACS)Cell groups sorting

based their surface markers (receptors)

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The principle of flow cytometry

The Flow cytometry is a laboratory method used for the quick and multiparametric examination of separated cells (ie. blood cells) . By the help of this method, the cell types of a mixed cell population can be analysed, counted, measured based on their phenotype and fuctional state.

In practice, by the help of a fluorocytometer in same time cell size, granularity, 4-6 different fluorescence signals can be measured. Thus more parameter can be obtained from a cell type.

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Cell sorting by FACS machine

Cells have been fluorescently tagged with either red or green antibodies, though not every cell expresses the epitope and therefore some are not tagged either color.

The final step is sorting the cells which is accomplished by electrical charge. The computer determines how the cells will be sorted before the drop forms at the end of the stream. As the drop forms, an electrical charge is applied to the stream and the newly formed drop will form with a charge. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes. Drops that contain no cells are sent into the waste tube. The end result is three tubes with pure subpopulations of cells. The number of cells is each tube is known and the level of fluorescence is also recorded for each cell.

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Sorting FACSCalibur machine,analysis by CellQuest program.

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Applied laser for excitation

Red diode laser (635 nm)

Blue Argon Laser (488 nm)

Violet laser (405 nm)

Emission detectors

FL1 515-545 nm

FL2 560-600 nm

FL3 610-660 nm

FL4: 670 nm <

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Size and granularity of human peripheral blood cells (FSC, SSC)

Lymphocytes

Granulocytes

Monocytes

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Helper and cytotoxic T cells in human peripheral blood

(cytotoxic) T lymphocytes

(helper) T lymphocytes

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NK cells in human peripheral blood

T lymphocytes

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