Lab #6: Immunohistochemistry (IHC) Practical Of Histopathology

Overview Imunohistochemistry (IHC) combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label.

Cont. The body's response to the introduction of a foreign agent, known as the immune response, results in the production of antibodies. Antibodies bind tightly and specifically to an "epitope" (one specific structure) on an "antigen" (foreign molecule or structure). Fab region Paratope + Epitope Ab-Ag complex

Cont. An antigen can be defined as "anything that can be bound by an antibody." This can be an enormous range of substances from simple chemicals, sugars, and small peptides to complex protein complexes such as a virus capsid. Not all antigens directly elicit an antibody response. Some require a carrier to be effective. These generally smaller antigens are called haptens.

Polyclonal Ag injected into host animal. Serum collected and purified. Multiple antibodies produced by different cell types bind multiple epitopes on Ag Monoclonal Ag injected into mouse. Lymphocytes isolated, hybridized. One antibody produced by one cell type binds one epitope on Ag.

Cont. Antibodies can be generated by injecting animals with antigens, and then collecting serum after the immune response has taken place. If the antibodies are labeled with an easily detectable molecule (a fluorescent dye, an enzyme, etc.), they become powerful detection reagents for the antigen. This system has been exploited to generate exceptionally specific and sensitive "stains" which are used in histology as well as other disciplines.

Cont. The basic process depends upon selecting an antibody sufficiently specific to bind an antigen in situ. The antibody/antigen conjugate is then identified using a variety of signal generating molecules triggered either by the antibody/antigen interaction or by secondary processes. Labels Enzyme labels: Horseradish peroxidase & alkaline phosphatase Colloidal metal labels Fluorescent labels Radiolabels(radioisotopes) Ferritin (electron microscopy)

Cont. Immunohistochemistry is generally carried out in sectioned tissue, which allows the antibodies free access to the interior of the cells. Immunohistochemistry can also be carried out on cells either in free solution or bound to membranes, or on monolayers of cultured cells. Intracellular Immunohistochemistry requires that the antibody to the target antigen be able to penetrate the cell membrane and whatever cell wall may be present before it can attach to the antigen. This requires a number of steps not required for sectioned tissue.

Cont. Primarily the cell membrane must be made permeable to the antibody, though at the same time the integrity of the cell contents and structures must be maintained. This is normally achieved through antigen retrieval techniques.

Methods for Ag detection Direct Indirect

Direct method The antibody against the macromolecule is labeled with(e.g a fluorescent dye). The antibody is then permitted to react with the macromolecule, and the resultant complex may be viewed with a fluorescent microscope. Indirect method a fluorescent labeled antibody is prepared against the primary antibody specific for the macromolecule of interest. forming a secondary complex visible by fluorescent microscopy

Cont. The indirect method is more sensitive than the direct method because numerous labeled anti-antibodies bind to the primary antibody, making them easier to visualize. In addition, the indirect method does not require labeling of the primary antibody(monoclonal), which often is available only in limited quantities.

Unmasking of antigen sites(antigen retrieval) Delayed fixation or poor fixation may cause loss of antigenicity or diffusion of antigens into the surrounding tissue. Antigens can be masked by the chemical processes involved in formalin fixation and paraffin processing and that some form of unmasking of these antigens is required . When formalin-based fixatives are used, intermolecular and intramolecular cross-linkages are formed with certain structural proteins. These are responsible for the masking of the tissue antigens.

Manual methods for antigen unmasking include: Proteolytic enzyme digestion (trypsin and protease):it is generally accepted that the digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are uncovered. Heat-mediated antigen retrieval techniques Microwave antigen retrieval Pressure cooker antigen retrieval Steamer Water bath (at 90°C) Autoclave Antigen retrieval solutions: high pH solutions (recommended for certain antibodies) or lower pH 6.0 for more general use.

Immunohistochemistry for the assessment of ER/PR, HER2 expression in breast cancer Immunohistochemistry (IHC) is used to define four breast cancer subtypes for expression of estrogen receptor (ER) or progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2): ER/PR+, Her2+ ER/PR+, Her2− (most common) ER/PR−, Her2+ ER/PR−, Her2−. About 80% of all breast cancers are “ER-positive.” That means the cancer cells grow in response to the hormone estrogen. About 65% of these are also “PR-positive.” They grow in response to another hormone, progesterone. The triple negative subtype (ER/PR−, Her2−) had the worst overall survival  The assessment of HER2 in human tumor cells has recently become important since the amplified gene is predictive of response to the novel humanized HER2 antibody Herceptin®/trastuzumab. Historically the major factors directing the appropriate treatment for breast cancer have been those regarding prognosis, such as indicators of how long the average patient is likely to survive. As a result, the relative aggressiveness of a tumor has been gauged and the appropriate treatment regime has been planned. HER2 is a member of the epidermal growth factor receptor (EGFR) family of molecules and is encoded for by the HER2 proto-oncogene on the long arm of chromosome 17. HER2 is overexpressed in 10–20% of primary breast cancers. Overexpression indicates poor prognosis. Breast cancers showing over expression are candidates for treatment with trastuzumab (Herceptin). Studies show that trastuzumab can reduce the risk of occurrence by one-half and mortality by one-third in early-stage breast cancer patients.

Tumors that are ER/PR-positive are much more likely to respond to hormone therapy than tumors that are ER/PR-negative. You may have hormone therapy after surgery, chemotherapy, and radiation are finished. These medications  taken help stop cancer from coming back by blocking hormone receptors. A class of medicines called aromatase inhibitors actually stops estrogen production. They’re only used in women who’ve already gone through menopause.

HER-2 In about 20% of breast cancers, the cells make too much of a protein known as HER2. These cancers tend to be aggressive and fast-growing. The standard of care to treat curable HER2-positive breast cancer has been to give chemotherapy with the HER2-targeted monoclonal antibody. 1 2 3 Formalin-fixed paraffin sections immunostained to demonstrate HER2 are examined microscopically and scored. It is recommended that formalin-fixed paraffin sections should be antigen retrieved using the water bath method and citrate buffer, pH 6.0, to ensure optimal immunostaining.

fluorescence in situ hybridization (FISH) for HER2 assessment The centromeric region of chromosome 17 is marked with a green fluorescent signal and the HER2 gene with a red signal.

General Immunohistochemistry Protocol Part 1:Tissue preparation 1. Fixation Fresh unfixed, fixed, or formalin fixation and paraffin embedding 2. Sectioning 3. Whole Mount Preparation

Proteolytic enzyme method and/or Heat-induced method Part 2: pretreatment 1. Antigen retrieval Proteolytic enzyme method and/or Heat-induced method 2. Inhibition of endogenous tissue components 3% H2O2, 0.01% avidin 3. Blocking of nonspecific sites 10% normal serum Part 3: staining Make a selection based on the type of specimen, the primary antibody, the degree of sensitivity and the processing time required.

Controls It is to test for a protocol or procedure used. Positive Control It is to test for a protocol or procedure used. It will be ideal to use the tissue of known positive as a control. Negative Control It is to test for the specificity of the antibody involved.

What cellular antigens can we target? Cytoplasmic Nuclear Cell membrane Lipids Proteins

Identify replicating cells Applications Identify replicating cells Locate cells that are signaling Locate apoptotic cells

Identify activation states Examine cytoskeletal structure Identify different types of cells in a tissue

Midterm Exam Sat., 3/11/2018 11:00 am Topics: 1. Introduction, 2. Tissue processing, 3. section cutting, 4. staining techniques, 5. immunohistochemistry