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== 1. Histology ==
Histology is the study of the microscopic structure of biological material and the ways in which individual components are structurally and functionally related. It is central to medical science since it stands at the crossroads between biochemistry, molecular biology and physiology on the one side, and pathologic processes that cause disease on the other. Although often thought of as an archaic discipline, practical knowledge of histology is in actuality an integral part of modern investigative techniques and current medical practice
In this laboratory manual we will focus on the basic structure of human tissues. We will concentrate on structure-function correlations that are important in the understanding of disease processes. Thus, we will not attempt to provide a comprehensive review of all structures in the body; instead we will focus just on the structural relationships that are integral to disease.
Almost all of the tissues we will review are human tissues obtained at autopsy or from surgical biopsies. As a general rule all fresh tissues are fixed in 10% neutral buffered formalin and are embedded in paraffin wax before cutting microscopic tissue sections. The embedding process requires dehydration of the tissues using organic solvents, permeation of the tissues with paraffin wax, and hardening of the wax for cutting. Tissue sections are then cut at 5 to 7 m in thickness and placed on glass slides. The tissues are then rehydrated and stained. This dehydration-wax embedding - rehydration cycle results in dissolution of any lipid materials within the tissues. This may lead to alterations in the morphology of tissues. However, if you understand the process you can overlook these artifacts and still make accurate assessments of the tissue. One classic “artifact” is the loss of fat from liver tissue obtained from a patient with fatty liver. This leaves holes in the tissue where the fat globules had been situated before they were dissolved away. These and other classic artifacts will become second nature to you as you review tissue sections.
== Overview of Tissue Preparation and Staining for Microscopy ==
1. '''Obtaining tissues''' - Human material is obtained at autopsy or from surgical biopsies.
2. '''Fixation''' - To preserve the tissue, it is placed immediately in a fixative which acts to preserve the cell and tissue constituents in as lifelike a manner as possible after death. In postmortem tissue, considerable autolysis may have occurred prior to fixation. Formalin (10%) is the fixative most often used by pathologists.
3. '''Dehydration''' - The fixed tissues must be dehydrated in order to embed them in paraffin for sectioning. Water is removed from the tissues by passing them through a series of increasingly concentrated solutions of alcohol.
4. '''Clearing''' - Absolute alcohol is not miscible with paraffin. Thus, the alcohol must be removed from the tissue and replaced with an agent that mixes with molten paraffin. The most commonly used clearing agent is xylene. The xylene makes the tissues translucent or “clears” them.
5. '''Embedding''' - Following clearing, the tissue is placed in the embedding agent, molten paraffin, and allowed to steep until the tissue is thoroughly infiltrated by the embedding medium. The preparation is then cooled, the paraffin solidifies, and the block of tissue can now be cut with a minimum of distortion. The paraffin infiltrates the interstices of the tissue and thus provides internal support as well as external support for sectioning.
6. '''Sectioning''' - The tissue is now cut into very thin slices, usually 5 to 7 m, with a microtome. The sections are then mounted on glass slides and stained.
7. '''Staining''' - For morphologic study, it is necessary to create color contrasts in the tissues by staining. Certain terms are used to distinguish the staining reaction of a cell. The term basophilic indicates that the structure can be stained with the basic dye hematoxylin. All nuclei are basophilic. Cytoplasmic elements may be either basophilic, acidophilic or, neutral. Eosin is the most commonly used acid stain and any acid components that stain positive with eosin are termed eosinophilic.
8. '''Other stains''' used in preparing slides – Most slides for histology and pathology are stained with Hematoxylin and Eosin (H&E). Additional staining techniques are utilized to demonstrate specific characteristics of tissues. In any staining process variations in the tissue and the technical procedure may lead to minor color modifications in individual slides, but, in general, the reactions are as stated below.
a. '''Masson’s Trichrome stain''' (hematoxylin, acid fuchsin, and aniline blue): nuclei stain black or dark blue; cytoplasm stains red by the acid fuchsin; reticular and collagen fibers stain blue with aniline blue.
b. Gomori’s Trichrome stain: Another version of a trichrome stain that stains nuclei - red-purple; normal muscle myofibrils - green-blue with distinct A and I bands; intermyofibrillar muscle membranes – red; and interstitial collagen - green
c. '''Periodic acid-Schiff’s reagent (PAS)'''. The PAS method stains glycogen, mucin, connective tissue fibers, and other structures that contain carbohydrates, pink, red, or maroon. The periodic acid converts adjacent 1, 2 glycol groups to aldehydes and the basic Fuchsin of Schiff’s reagent stains the aldehydes. Sometimes Hematoxylin is used as a counter stain giving you a PASH.
c. '''Silver stain'''. This special procedure employs silver nitrate to specifically demonstrate reticular fibers, neurofibrils of neurons and granules in enteroendocrine cells. These structures are stained black whereas other tissue components may take on a faint gray background stain without revealing detail.
d. '''Toluidine blue'''. Used to demonstrate granules in mast cells. Nuclei are deep blue; mast cell granules are reddish-purple.
e. '''Verhoeff-Van Gieson stain (VVG)'''. This method is used for identifying elastic fibers in tissues such as skin, aorta, etc. The elastic fibers will be stained blue-black and background will be stained yellow.
Histology is the study of the microscopic structure of biological material and the ways in which individual components are structurally and functionally related. It is central to medical science since it stands at the crossroads between biochemistry, molecular biology and physiology on the one side, and pathologic processes that cause disease on the other. Although often thought of as an archaic discipline, practical knowledge of histology is in actuality an integral part of modern investigative techniques and current medical practice
In this laboratory manual we will focus on the basic structure of human tissues. We will concentrate on structure-function correlations that are important in the understanding of disease processes. Thus, we will not attempt to provide a comprehensive review of all structures in the body; instead we will focus just on the structural relationships that are integral to disease.
Almost all of the tissues we will review are human tissues obtained at autopsy or from surgical biopsies. As a general rule all fresh tissues are fixed in 10% neutral buffered formalin and are embedded in paraffin wax before cutting microscopic tissue sections. The embedding process requires dehydration of the tissues using organic solvents, permeation of the tissues with paraffin wax, and hardening of the wax for cutting. Tissue sections are then cut at 5 to 7 m in thickness and placed on glass slides. The tissues are then rehydrated and stained. This dehydration-wax embedding - rehydration cycle results in dissolution of any lipid materials within the tissues. This may lead to alterations in the morphology of tissues. However, if you understand the process you can overlook these artifacts and still make accurate assessments of the tissue. One classic “artifact” is the loss of fat from liver tissue obtained from a patient with fatty liver. This leaves holes in the tissue where the fat globules had been situated before they were dissolved away. These and other classic artifacts will become second nature to you as you review tissue sections.
== Overview of Tissue Preparation and Staining for Microscopy ==
1. '''Obtaining tissues''' - Human material is obtained at autopsy or from surgical biopsies.
2. '''Fixation''' - To preserve the tissue, it is placed immediately in a fixative which acts to preserve the cell and tissue constituents in as lifelike a manner as possible after death. In postmortem tissue, considerable autolysis may have occurred prior to fixation. Formalin (10%) is the fixative most often used by pathologists.
3. '''Dehydration''' - The fixed tissues must be dehydrated in order to embed them in paraffin for sectioning. Water is removed from the tissues by passing them through a series of increasingly concentrated solutions of alcohol.
4. '''Clearing''' - Absolute alcohol is not miscible with paraffin. Thus, the alcohol must be removed from the tissue and replaced with an agent that mixes with molten paraffin. The most commonly used clearing agent is xylene. The xylene makes the tissues translucent or “clears” them.
5. '''Embedding''' - Following clearing, the tissue is placed in the embedding agent, molten paraffin, and allowed to steep until the tissue is thoroughly infiltrated by the embedding medium. The preparation is then cooled, the paraffin solidifies, and the block of tissue can now be cut with a minimum of distortion. The paraffin infiltrates the interstices of the tissue and thus provides internal support as well as external support for sectioning.
6. '''Sectioning''' - The tissue is now cut into very thin slices, usually 5 to 7 m, with a microtome. The sections are then mounted on glass slides and stained.
7. '''Staining''' - For morphologic study, it is necessary to create color contrasts in the tissues by staining. Certain terms are used to distinguish the staining reaction of a cell. The term basophilic indicates that the structure can be stained with the basic dye hematoxylin. All nuclei are basophilic. Cytoplasmic elements may be either basophilic, acidophilic or, neutral. Eosin is the most commonly used acid stain and any acid components that stain positive with eosin are termed eosinophilic.
8. '''Other stains''' used in preparing slides – Most slides for histology and pathology are stained with Hematoxylin and Eosin (H&E). Additional staining techniques are utilized to demonstrate specific characteristics of tissues. In any staining process variations in the tissue and the technical procedure may lead to minor color modifications in individual slides, but, in general, the reactions are as stated below.
a. '''Masson’s Trichrome stain''' (hematoxylin, acid fuchsin, and aniline blue): nuclei stain black or dark blue; cytoplasm stains red by the acid fuchsin; reticular and collagen fibers stain blue with aniline blue.
b. Gomori’s Trichrome stain: Another version of a trichrome stain that stains nuclei - red-purple; normal muscle myofibrils - green-blue with distinct A and I bands; intermyofibrillar muscle membranes – red; and interstitial collagen - green
c. '''Periodic acid-Schiff’s reagent (PAS)'''. The PAS method stains glycogen, mucin, connective tissue fibers, and other structures that contain carbohydrates, pink, red, or maroon. The periodic acid converts adjacent 1, 2 glycol groups to aldehydes and the basic Fuchsin of Schiff’s reagent stains the aldehydes. Sometimes Hematoxylin is used as a counter stain giving you a PASH.
c. '''Silver stain'''. This special procedure employs silver nitrate to specifically demonstrate reticular fibers, neurofibrils of neurons and granules in enteroendocrine cells. These structures are stained black whereas other tissue components may take on a faint gray background stain without revealing detail.
d. '''Toluidine blue'''. Used to demonstrate granules in mast cells. Nuclei are deep blue; mast cell granules are reddish-purple.
e. '''Verhoeff-Van Gieson stain (VVG)'''. This method is used for identifying elastic fibers in tissues such as skin, aorta, etc. The elastic fibers will be stained blue-black and background will be stained yellow.