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MCB - Histo Methods

Hisotological Methods and Microscopy

resolution ability to distinguish between 2 points (differs from magnification which just enlargens objects)
electron microscope vs light microscope Light uses a light beam (longer wavelength) with a glass lenses and has less resolution Electron uses an electron beam (shorter wavelength) and an electromagnet lenses for higher resolution
Confocal (bright-field) microscope illuminates: melanin, hemoglobin, and cholorphyll, and mvmt of structures (cilia ex) Mechanism: molecules and stains absorb and diffract light
Phase Contrast microscope Illuminates: mitochondria, lysosomes, chromosomes, nucleoli Mechanism: sample alters amplitude and phase relationship of light
Differential Interference Contrast (DIC) microscope Illumination: cell and organelle edges, Fiber-like structures (mm, bone, nerve) Mechanism: structures rotate plane of light)
Describe how flourescence can be used to understand structure and pathophys. of cells and tissues? Different molecules will autoflouress when light of certain wavelength is shown on them. This allows us to see what molecules are in structures as well as if a pathological molecule is present (ex: Melanin and lipfusion ex)
acidophilic positively charged molecule that an acidic dye binds to
basophilic negatively charged molecule that a basic dye binds to
acidic dye Dye is negatively charged, binds acidiophilic subtrates such as secretory granules, mitochondria, and SER.
Eosin dye acidic dye that reacts with primary amines
basic dye positively charged dye that reacts with basophilic substrates use all nucleic acids (Chromatin DNA, Nucleolus RNA, ribosomal RNA)
hematoxylin dye basic dye that use Al as a mordant to bind nucleic acids
alican blue Dye basic dye that binds glycosaminoglycans (GAGs), aka proteoglycans
PAS dye method uses a schiff reagent to bind to carbohydrates (such as those found in mucous)
Feulgen dye method uses acid to break bonds and a schiff reagent to bind DNA
How can enzyme histo be used to enhance structural information? combine with electron microscopy to determine organelle enzyme is expressed in (note: sample prep will limit structural info though)
Immunohistochemistry using antibodies to locate specific molecules mainly proteins and some carbohydrates
2 types of immunohistochem. direct - labeled antibody (ab) binds antigen indirect - unlabeled ab binds antigen and a labeled ab binds the unlabeled to allow for greater sensitivity and amplification
some limitations of immunohistochem. antigenicity (how reactive protein is to ab), antigen synthesis, and ab's are not a renewable source
How can immunohistochem be used clinically can be used to detect proteins characteristic of diseased cells, carbs of certain bacteria, carbs that cause blood type, or carbs seen on carcinomas
in situ hybridization uses a dig-labeled DNA or RNA probe to bind to a complementary segement of DNA. An ab coulpled to an alkaline phosphotase binds to dig. subtrate that changes color when phosphate is remove is added to localize sequence
General fxn for (1) LM and EM microscopes, (2) acid/base staining, (3) histochem, (4) Immunohistochem. and In situ hybridization (1) see details of cells, tissues, organs (2) creates contrast to see details with microscopes (3) correlate molecular and structural details of cells, tissues, organs (4) locate specific molecules in cells/tissues/organs
Created by: c.phill