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Lecture

1. Histological Techniques.pdf

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Department
Anatomy & Cell Biology
Course
ANAT 261
Professor
Craig Mandato
Semester
Fall

Description
Cross Section Oblique Section Longitudinal Section Cross Section Oblique Section Longitudinal SectionNaveen Sooknanan McGill Fall 2011 Histological Techniques: Histology can be divided into two important categories:  Medical histology is the identification of tissues and cells from microscopic slides  Dynamic histology is an extension where, along with the identification of these tissues, we examine the functions of the cells and molecules that make up the tissue Systemic human anatomy involves observations at the macroscopic level with the eyes only, while histology takes place at the microscopic level with the use of sections and slides.  These sections can be made up of various tissue combinations including epithelial, muscular, connective and nervous tissue o It is the different composition of these tissues that defines each particular section Take the following cross section of an arm, for example:  The skin is an organ made up of epithelial and connective tissue  The fascia, tendons and aponeurosis are structures made of connective tissue  The muscle is an organ made of muscular tissue  The bone is an organ and tissue made of connective tissue  Blood vessels are organs made of epithelial, connective and muscle tissue  The nerves are a tissue, organ and system made of nervous tissue The diagram above represents the various planes of section in which a tubular structure can be cut  A cut perpendicular to the length of the tube produces a cross section  A cut parallel to the tube produces a longitudinal section  Anything between these produces an oblique section o Most sections are oblique, it is very difficult to get a perfect cross section of a tissue Various units of measurements of length are used in histology:  1cm = 10mm  1mm = 1000μm (microns)  1μm = 1000nm  1nm = 10A (Angstrom units) This is useful because:  The thickness of a section for a light microscope is 5μm  The diameter of a cell is 10μm  Diameter of a lysosome is 200nm  Thickness of the plasma membrane is 75A (or 7.5nm) 1Naveen Sooknanan McGill Fall 2011 In order to produce a histological slide, various steps must be carried out in order to have a proper preservation and staining of the desires tissues. For our purposes, slides are usually takes from mouse specimens. The sample must first be fixed in order to preserve the tissues of the sample  For light microscopy, 2 techniques may be used: perfusion and immersion o Immersion involves placing the sample in a large amount of fixative and allowing penetration from the outside in. This method takes a long time and can cause degradation of the inner tissue before the fixative reaches the center of the sample o Perfusion involves the injection of the fixative into a living sample and allowing blood circulation to pump the fixative into the tissue from the inside out. The method it much faster and prevents tissue degradation o Common fixatives for LM are formalin, mercuric chloride (poison), picric acid and Bouin’s fluid (acetic acid, picric acid, formalin (dilute formaldehyde) and water)  Picric acid is rarely used alone as it is highly explosive when it’s dry  Bouin’s fluid is preferred because it is safer  For electron microscopy, perfusion is a preferable method of fixation to preserve subcellular structures which are visible under the EM o There is a double fixation process involving 2-5-5% glutaraldehyde and osmium tetroxide (OsO 4  These fixatives work much faster than LM fixatives  Another method of fixation is the freezing of the sample and cutting into slices called cryosection o This freezing is done with liquid nitrogen The fixed sample must then be dehydrated with ethanol for both LM and EM  Starting with a 50/50 ethanol/water solution and working gradually down to a 100% ethanol solution, water slowly diffuses out of the tissue o This is done in solutions of ethanol and water changing in increments of 10%  Ethanol diffuses fat cells during dehydration and they can’t be seen on histological slides, leaving “ghost cell” pockets o This can be especially seen in the hypodermis of the skin In a process called clearing, the ethanol is removed from the sample using xylene which readies it a paraffin or epoxy solvent in the next step The tissue is the
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