Tissue Preparation Steps: Sectioning to Staining PDF
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Uploaded by SatisfactoryMagic765
Faculty of Medicine and Health Sciences
2021
Dr. Mustafa Ghanim & Dr. Fatina Hanbali
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Summary
This document details the different steps involved in tissue preparation, from sectioning to staining, in a histology course. It covers various techniques such as sectioning, staining with hematoxylin and eosin, and methods involving metal impregnation. The document also explores the medical applications of these techniques.
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Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 29/01/2021 1 Sectioning The hardened block with tissue and surrounding embedding medium is trimmed and...
Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 29/01/2021 1 Sectioning The hardened block with tissue and surrounding embedding medium is trimmed and placed for sectioning in an instrument called a microtome (see Figure 1–1b) Paraffin sections are typically cut at 3-10 µm thickness for light microscopy, but electron microscopy requires sections less than 1 µm thick 29/01/2021 2 29/01/2021 3 Medical application 29/01/2021 4 Staining Most cells and extracellular material are colorless, and to be studied microscopically tissue sections must be stained (dyed) Methods of staining make various tissue components not only conspicuous but also distinguishable from one another 29/01/2021 5 Cont. Staining Dyes stain material more or less selectively, often behaving like acidic or basic compounds and forming electrostatic (salt) linkages with ionizable radicals of macromolecules in tissues Cell components, such as nucleic acids with a net negative charge (anionic), have an affinity for basic dyes and are termed basophilic cationic components, such as proteins with many ionized amino groups, stain more readily with acidic dyes and are termed acidophilic 29/01/2021 6 Basic dyes Basic dyes include toluidine blue, alcian blue, and methylene blue Hematoxylin behaves like a basic dye, staining basophilic components The tissue components that ionize and react with basic dyes have acids in their composition (DNA, RNA, and glycosaminoglycans) 29/01/2021 7 Acidic dyes Acid dyes (eg, eosin, orange G, and acid fuchsin) stain the acidophilic components of tissues such as mitochondria, secretory granules, and collagen 29/01/2021 8 Hematoxylin & Eosin The simple combination of hematoxylin and eosin (H&E) is the most commonly used stain Hematoxylin stains DNA in the cell nucleus, RNA-rich portions of the cytoplasm, and the matrix of cartilage, producing a dark blue or purple color In contrast, eosin stains other cytoplasmic structures and collagen pink (Figure 1–2a) 29/01/2021 9 Figure 1–2a 29/01/2021 10 Cont. Hematoxylin & Eosin In H&E, eosin is considered a counterstain, which is usually a single dye applied separately to distinguish additional features of a tissue Trichrome stains (eg, Masson’s trichrome), allow greater distinctions among various extracellular tissue components 29/01/2021 11 PAS The periodic acid–Schiff (PAS) reaction utilizes the hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules distinctly purple or magenta Figure 1–2b shows an example of cells with carbohydrate-rich areas well-stained by the PAS reaction The DNA of cell nuclei can be specifically stained using a modification of the PAS procedure called the Feulgen reaction 29/01/2021 12 Figure 1–2b 29/01/2021 13 Pretreatment of a tissue section Basophilic or PAS-positive material can be further identified by enzyme digestion, pretreatment of a tissue section with an enzyme that specifically digests one substrate For example, pretreatment with ribonuclease will greatly reduce cytoplasmic basophilia with little overall effect on the nucleus, indicating the importance of RNA for the cytoplasmic staining 29/01/2021 14 Studying lipid-rich structures Lipid-rich structures are revealed by avoiding the processing steps that remove lipids, as treatment with heat and organic solvents and staining with lipid-soluble dyes such as Sudan black Used in the diagnosis of metabolic diseases involving intracellular accumulations of cholesterol, phospholipids, or glycolipids 29/01/2021 15 Metal impregnation Less common methods of staining employ metal impregnation Using solutions of silver salts to visual certain ECM fibers and specific cellular elements in nervous tissue 29/01/2021 16 Time needed for sample preparation Slide preparation, from tissue fixation to observation with a light microscope, may take from 12 hours to 2½ days, depending on the size of the tissue, the embedding medium, and the staining method The final step before microscopic observation is mounting a protective glass coverslip on the slide with clear adhesive 29/01/2021 17