Immunochemistry
The process of localizing proteins in cells of a tissue section using the principle of antigens in tissue binding to their respective antibodies is known as Immunohistochemistry (IHC). This technique is widely used for the diagnosis of cancer. The antibodies are tagged with color-producing dyes to make them easily visible. Horseradish peroxidase and phosphatase are the two commonly used color-producing tags. Different fluorophores like FITC are also used to tag the antibodies as an alternate method. This method is widely used in confocal laser scanning microscopy to visualize two interacting protein molecules. Adinocarcinomas, Hodgkin's disease, yolk sac tumors and hepatocellular carcinoma,, Gastrointestinal Stromal Tumors (GIST) and prostrate cancer are diagnosed by conducting immunohistochemistry using various antigens like Carcinoembryonic antigen, CD 15 & CD30 or Alpha fetoprotein or CD117 or prostrate specific antigen accordingly. Direct and indirect methods are the two methods used for the immunohistochemical staining of antigens.
In the direct method, one labelled antibody is used for staining. The antibody directly binds to the antigen which is being stained. Involving only one labeled antibody ( FITC conjugated antiserum), this direct method is also known as one-step staining method. Though this method is quick since it uses only one antibody, it is rarely used after the indirect method was introduced.
In the indirect method of immuno-histochemical staining, one antibody is used against the antigen which is being examined and a second labelled antibody is used against the first. The unlabelled primary antibody or the first layer reacts with tissue antigen and the second layer or the labelled secondary antibody reacts with the primary antibody. This indirect method is considered more sensitive, since there is good signal amplification noticed through many secondary antibody reactions with various antigenic sites on the primary antibody.
Histopathology
Histopathology involves study of diseased tissues thereby aiding diagnosis of tumors. Histopathological examination of tissues involves three stages - surgery, biopsy or autopsy.
After collecting the diseased tissues, they are first stabilized by placing in a fixative, to prevent decay. Formalin (10% formaldehyde in water) is the fixative used normally.
- The stabilized sample tissues are first transferred to a container where permitted reagents are allowed to act on the tissues.
- Concentrated ethanol helps to dehydrate the tissues. Toluene or Xylene is used after the above procedure to immerse the tissues followed by hot liquid paraffin wax. This process takes 12 to 16 hours, during which paraffin replaces the water in the tissue turning it soft and moist. Now the sample is ready for embedding.
- The tissue is transferred and set in a mold. During this process of embedding, additional paraffin is added to prepare a hard paraffin block.
- Microtome is used to section the embedded tissues into very thin sections of about 3-7 micrometer in thickness. These thin layers or sections are now ready for microscopic study.
- One or more pigments are used to stain the sections to view the details clearly. A combination of hematoxylin and eosin is used to stain the tissues. The cytoplasm pink is got by using eosin and nuclei blue by hematoxylin. Saffron, silver salts and some artificial dyes are the other compounds used to color tissue sections. For staining specific proteins, carbohydrates and liquids, antibodies are also used. The technique known as immunohistochemistry has helped scientists in the microscopic studies to identify different categories of cells specifically.
- A medically qualified expert or pathologist examines the histological slides microscopically.
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