Module 1: Pt.2

• Primary cell cultures - dispersed mechanically or enzymatically from a tissue or organ and placed with sterile procedures in a clear dish to which they adhere, usually as a single layer
• Some cells can be maintained in vitro for long periods because they become immortalized and constitute a permanent cell line
• Transformation - process that promote cell immortality and are similar to the initial changes in a normal cells becoming a cancer cell
• Enzyme histochemistry (cytochemistry) - localizing cellular structures using a specific enzymatic activity present in those structure.
• For enzyme histochemistry:

  (1) tissue sections are immersed in a buffer containing the substrate of the enzyme to be localized at the appropriate temperature and pH;

  (2) the enzyme is allowed to act on its substrate;

  (3) the section is then put in contact with a marker compound that reacts with a product of the enzymatic action on the substrate; and (4) the final product from the marker, which must be insoluble and visible by light or electron microscopy, precipitates over the site of the enzymes, identifying their location
• Examples of enzymes that can be detected histochemically include the following:

  • Phosphatases - which remove phosphate groups from macromolecules
• Examples of enzymes that can be detected histochemically include the following:

  • Dehydrogenases - which transfer hydrogen ions from one substrate to another, such as many enzymes of the citric acid (Krebs) cycle, allowing histochemical identification of such enzymes in mitochondria
• Examples of enzymes that can be detected histochemically include the following:

  • Peroxidase, which promotes the oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide
• specific macromolecule - may also be identified by using tagged compounds or macromolecules that bind specifically with the molecule of interest
• Examples of molecules that interact specifically with other molecules include the following:

  • Phalloidin - compound extracted from mushroom, Amanita phalloides, interacts strongly with the actin protein of microfilaments
• Examples of molecules that interact specifically with other molecules include the following:

  • Protein A - purified from Staphylococcus aureus bacteria, binds to Fc region of antibody molecules, and can be used to localize naturally occurring or applied antibodies bound to cell structures
• Examples of molecules that interact specifically with other molecules include the following:

  • Lectins - glycoproteins derived mainly from plant seeds, bind to carbohydrates with high affinity and specificity
• Immunohistochemistry - routinely used labeled antibodies to to identify and localize many specific proteins, not just those with enzymatic activity
• The body’s immune cells interact with and produce antibodies against other macromolecules (antigen)
• antigens - are recognized as “foreign,” not a normal part of the organism, and potentially dangerous.
• Antibodies - belong to the immunoglobulin family of glycoproteins and are secreted by lymphocytes.
• Polyclonal antibodies - derived from different groups (clones) of lymphocytes in injected animal recognize different parts of protein x and each clone produces an antibody against that part. These antibodies are collected from the animal's plasma, resulting in a mixture of antibodies targeting various regions of the antigen.
• Advantage of using a monoclonal antibody rather than polyclonal antibodies is that it can be selected to be highly specific and to bind strongly to the protein to be detected, with less nonspecific binding to other proteins that are similar to the one of interest.
• Monoclonal antibodies - produced by isolating activated lymphocytes from an animal (e.g., mouse) that has been injected with the antigen. These lymphocytes are fused with tumor cells to create hybridoma cells, which can produce identical antibodies targeting a specific part of the antigen.
• direct method of immunocytochemistry - utilizes a labeled antibody that binds the protein of interest
• Indirect immunohistochemistry - involves sequential application of two antibodies and additional washing steps
• secondary antibody - is produced in an animal species different (foreign) from the one that produced the primary antibody
• Hybridization - is the process of specific binding between two single strands of nucleic acid, occurring when the strands are complementary.

  This binding is influenced by the similarity of their nucleotide sequences, allowing for specific identification of sequences in genes or RNA under stringent conditions.
• Examples of specific antigens with diagnostic importance.

  
• Artifact - a minor structural abnormality that can occur in tissues as a result of certain steps in a procedure
• Structure’s three-dimensional volume is cut into very thin sections, the sections appear microscopically to have only two dimensions: length and width.