Basic techniques in histology, histochemistry, immunohistochemistry, cell, organelles, plasma membrane, glycocalyx, transmembrane proteins, movement across membrane, sodium pump, Cl- channel, cystic fibrosis, mitochondria, endoplasmic reticulum

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Department
Biomedical Science
Course
BMS 460
Professor
D.Rao Veeramachaneni
Semester
Fall

Description
30 August Basic Techniques in Histology Tissue processing Fixation, embedding, sectioning, and staining Autopsy (human)/necropsy (animal) Take sample of tissue Biopsy – small sample Smears (e.g. pap, oral) Incisional Embedding media: paraffin or plastic Stains: Hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), Toluidine blue, Trichrome etc. Hematoxycin (basic) – nucleus (acidic pH, basophilic) Eosin (acidic) – cytoplasm (basic, aciophilic/eosinophilic) Periodic acid-Schiff picks up on glycogen (sugars) Evaluation Microscopy Light microscopy (bright-field, contrast, fluorescence) Electron microscopy (transmission, scanning) A major difference among these types of microscopy is their resolving power (resolution) ~0.2 μm or 200 nm with LM vs. 1 to 2 nm with TEM An example of histological description/functional correlates Know terms A hepatocyte has a round, euchromatic basophilic nucleus and a mainly eosinophilic cytoplasm with patches of basophilia because of the presence of rough endoplasmic reticulum. The highly euchromatic nucleus indicate that these simple-appearing cells are engaged in a multiplicity of highly complex functions: large amounts of dispersed nuclear chromatin point to the transcription of a large variety of genes. Interpretation and understanding the three-dimensional structure and organization of tissues and organs A major challenge that confronts the student in histology is how to interpret and understand the three-dimensional structure and organization of tissues and organs by looking at apparently random two-dimensional slices of these structures. In a clinical setting, biopsy segments are typically much smaller, perhaps a few cubic millimeters and thus it makes interpretation more difficult. An accurate description of the appearance, organization, and composition of the cells, tissues, and organs is the key for understanding their function as well as abnormal changes that may occur An example: glandular structures The structure of exocrine glands ranges from the unicellular goblet cell to complex exocrine glands. Salivary glands Glands that produce secretions by both exocrine and endocrine mechanisms Pancreas (a compound acinar exocrine gland with clusters [islets] of endocrine cells) Liver (a branched lobar/lobular gland in which the parenchymal cells [hepatocytes] secrete an exocrine product [bile] into a system of ducts and endocrine products [e.g., serum proteins] into the bloodstream Histochemistry Various stains can be used in combination to identify unique cell types, e.g., pulmonary neuroendocrine cells (PNECs that give rise to carcinoid tumors) can be seen in the section stained with toluidine blue (TB) and basic fuchsin (BF) but not in hematoxylin- eosin (H&E)-stained section Whereas eosin does not stain PNEC or goblet cell secretions, note eosinophilic secretory granules in Paneth cells in crypt of Lieberkuhn in jejunum Biochemistry Biopsy material submitted for diagnosis are often worked up on fresh, frozen sections for histochemistry, biochemistry and molecular genetics Frozen sections: skeletal muscle biopsies Mitochondrial stain: Gomori’s rapid trichrome ATPase reaction: eosin counterstain Oxidative enzyme reactions: NADH-TR Glycogen content: PAS Immunohistochemistry Fluorescence in situ hybridization, FISH Often used when evaluating certain cancers to determine what type of cell the cancer originated from based on the type of antigens the cells express Labeled antibodies are applied to tissue sections; they bind if the targeted antigen is expressed by cells. The antigen-antibody complexes are observed under a fluorescence microscope or are developed into a colored stain that is visible under the bright-field microscope. The Cell and its Organelles Cell, the functional unit, is the target of injury Organelles as targets of injury Plasma membrane Endoplasmic reticulum Golgi apparatus Mitochondria Lysosomes Cytoskeleton Examples of select diseases as target topics for each organelle Plasma Membrane and Glycocalyx A variety of proteins embedded or dissolved in the lipid bilayer perform most of the membrane’s function and, in turn, regulate cell function Trans-membrane proteins Transport Signal transduction Peripheral proteins Structural The extracellular domain of a plasma membrane is generally glycosylated by the CHO portions of the glycolipids and transmembrane glycoproteins Glycocalyx Protection and lubrication Cell-cell interaction; homing Contains enzymes Transmembrane Proteins Three major types of cell surface receptor proteins Ion channel-linked receptor Enzyme-linked receptor G-protein-linked receptor Movement of Molecules and Ions Across Membranes Various transport proteins mediate the passage Passive transport: movement of molecules or ions across the membrane down their concentration or electrochemical gradient; no energy required Active transport: protein-mediated movement of molecules or ions across the membrane against their concentration or electrochemical gradient; energy required Primary active transport: transport of a substance across membrane directly coupled to ATP hydrolysis Secondary active transport: simultaneous movement of two substances across the cell membrane indirectly coupled to ATP hydrolysis Cotransport (symport): both substances transported in same direction
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