Histology I PDF: Tissue Preparation Steps

Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 25/01/2021 1  Introduction Histology is the study of the tissues of the body and how these tissues are arr...

Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 25/01/2021 1  Introduction Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs This subject involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ 25/01/2021 2  Cont. Introduction The small size of cells and matrix components makes histology dependent on microscopes and molecular methods of study Advances in biochemistry, molecular biology, physiology, immunology, and pathology are essential for a better knowledge of tissue biology Familiarity with the tools and methods of any branch of science is essential for a proper understanding of the subject 25/01/2021 3  PREPARATION OF TISSUES FOR STUDY The preparation of tissue slices or “sections” that can be examined visually with transmitted light Because most tissues and organs are too thick for light to pass through, thin translucent sections are cut from them and placed on glass slides for microscopic examination of the internal structures 25/01/2021 4  Cont. PREPARATION OF TISSUES FOR STUDY The ideal microscopic preparation is preserved so that the tissue on the slide has the same structural features it had in the body This is often not feasible because the preparation process can remove cellular lipid, with slight distortions of cell structure 25/01/2021 5  Steps of tissue preparation The basic steps used in tissue preparation for light microscopy are shown in Figure 1–1(a) 25/01/2021 6  Cont. Steps of tissue preparation 25/01/2021 7  Fixation To preserve tissue structure and prevent degradation by enzymes released from the cells or microorganisms Pieces of organs are placed as soon as possible after removal from the body in solutions of stabilizing or cross-linking compounds called fixatives 25/01/2021 8  Cont. Fixation Because a fixative must fully diffuse through the tissues to preserve all cells, tissues are usually cut into small fragments before fixation to facilitate penetration To improve cell preservation in large organs, fixatives are often introduced via blood vessels, with vascular perfusion allowing fixation rapidly throughout the tissues 25/01/2021 9  Cont. Fixation One widely used fixative for light microscopy is formalin, a buffered isotonic solution of 37% formaldehyde Both this compound and glutaraldehyde, a fixative used for electron microscopy, react with the amine groups (NH2 ) of proteins, preventing their degradation by common proteases Glutaraldehyde also cross-links adjacent proteins, reinforcing cell and ECM structures 25/01/2021 10  Cont. Fixation Electron microscopy provides much greater magnification and resolution of very small cellular structures, and fixation must be done very carefully to preserve additional “ultrastructural” detail Typically in such studies, glutaraldehyde-treated tissue is then immersed in buffered osmium tetroxide, which preserves (and stains) cellular lipids as well as proteins 25/01/2021 11  Dehydration The fixed tissue must undergo dehydration by having its water extracted gradually by transfers through a series of increasing ethanol solutions, ending in 100% ethanol 25/01/2021 12  Clearing The ethanol is then replaced by an organic solvent miscible with both alcohol and the embedding medium a step referred to as clearing because infiltration with the reagents used here gives the tissue a translucent appearance 25/01/2021 13  Infliltration and embedding To permit thin sectioning, fixed tissues are infiltrated and embedded in a material that imparts a firm consistency Embedding materials include paraffin, used routinely for light microscopy, and plastic resins, which are adapted for both light and electron microscopy 25/01/2021 14  Cont. Infliltration and embedding The fully cleared tissue is then placed in melted paraffin in an oven at 52°-60°C, which evaporates the clearing solvent and promotes infiltration of the tissue with paraffin And then embedded by allowing it to harden in a small container of paraffin at room temperature Tissues to be embedded with plastic resin are also dehydrated in ethanol and then infiltrated with plastic solvents that harden when cross-linking polymerizers are added Plastic embedding avoids the higher needed with paraffin, which helps avoid tissue distortion temperatures 25/01/2021 15  Sectioning The hardened block with tissue and surrounding embedding medium is trimmed and placed for sectioning in an instrument called a microtome Figure 1–1(b) Paraffin sections are typically cut at 3-10 µm thickness for LM, but EM requires sections less than 1 µm thick 25/01/2021 16