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Questions and Answers
What is the primary function of chemical fixatives in tissue preparation?
What is the purpose of dehydration and clearing in tissue preparation?
What type of charge do cell substances like DNA and RNA have, and how do they react with stains?
What is the purpose of mounting tissue sections on glass slides?
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What type of substances react with eosin and other acidic stains?
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What is the most commonly used staining method in tissue preparation?
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What is the primary advantage of fluorescence microscopy over bright-field microscopy?
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Which microscopy technique uses the differences in refractive index of various natural cell and tissue components to produce an image?
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What is the end result of confocal microscopy?
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What is the purpose of autoradiography in microscopy?
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What type of light is used in fluorescence microscopy?
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What is the most commonly used microscopy method by students and pathologists?
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What is a common application of autoradiography in microscopy?
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What type of microscopy is commonly used to examine living cells in vitro?
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What is a common limitation of using fixation and paraffin embedding in histochemical techniques?
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Which of the following enzyme classes is commonly studied using histochemical techniques?
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What is a common application of cryostat sectioning in histochemical techniques?
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What is the primary purpose of immunohistochemistry?
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What is the difference between direct and indirect immunohistochemistry?
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What is the advantage of using indirect immunohistochemistry?
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What is in situ hybridization (ISH) used for?
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What is the role of the labeled primary antibody in direct immunohistochemistry?
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What type of label is often used in immunohistochemistry for light microscopy?
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What is the primary consideration when interpreting tissue sections?
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What is a common issue in tissue processing and slide preparation that can lead to misinterpretation?
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What is the significance of recognizing artifacts in tissue sections?
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Why is it important to consider the 2D representation of 3D structures in tissue sections?
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What is the consequence of not recognizing artifacts in tissue sections?
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Study Notes
Preparation of Tissues for Study
- Chemical fixatives, such as formalin, are used to preserve tissue structure by:
- Cross-linking proteins
- Denaturing proteins
- Inactivating enzymes
- Preventing cell autolysis or self-digestion
- Dehydration of fixed tissue in alcohol and clearing in organic solvents prepares it for:
- Embedding and sectioning
- Embedding in:
- Paraffin wax
- Epoxy resin allows the tissue to be cut into very thin sections (slices) with a microtome
- Sections are mounted on glass slides for:
- Staining
- Revealing specific cellular and tissue components with the microscope
- The most commonly used staining method is a combination of:
- H&E (Hematoxylin and Eosin) stains
- H&E act as basic and acidic dyes, respectively
- Cell substances with a net negative (anionic) charge, such as:
- DNA
- RNA reaction strongly with:
- Hematoxylin
- Basic stains and are said to be “basophilic”
- Cationic substances, such as:
- Collagen
- Many cytoplasmic proteins reaction with:
- Eosin
- Other acidic stains and are said to be “acidophilic”
Light Microscopy Techniques
- Bright-field microscopy uses ordinary light and tissue staining to produce colors, making it a common method used by students and pathologists.
- Fluorescence microscopy utilizes UV light, making only fluorescent molecules visible, and allows for the localization of specific fluorescent probes.
- Phase-contrast microscopy produces an image without staining by exploiting differences in refractive index of natural cell and tissue components, enabling observation of living cells.
- Confocal microscopy creates a 3D reconstruction from images by scanning a specimen at successive focal planes with a focused light beam, often from a laser.
Autoradiography
- Autoradiography is a process that localizes cell components synthesized using radioactive precursors.
- It detects silver grains produced by weakly emitted radiation in a photographic emulsion coating the tissue section or cells.
Autoradiography
- Enables studies of processes like tissue growth and cellular pathways of macromolecular synthesis
- Uses radioactive DNA precursors
Cell and Tissue Culture
- Cells can be grown in vitro from primary cultures (newly explanted tissues) or established cell lines
- Living cells can be examined using phase-contrast light microscopy
Enzyme Histochemistry
- Uses specific enzymatic activities to produce visible products in specific enzyme locations
- Typically uses frozen tissue sections, obtained with a cryostat
- Fixation and paraffin embedding denatures most enzymes
- Useful for studying enzyme classes like phosphatases, dehydrogenases, and peroxidases
- Peroxidase is often conjugated to antibodies used in immunohistochemistry
Visualizing Specific Molecules
- Some substances can specifically bind to certain targets in cells.
- Immunohistochemistry is based on specific reactions between an antigen and antibodies labeled with visible markers.
Types of Immunohistochemistry
- Direct Immunohistochemistry: uses a labeled primary antibody that directly binds to the antigen of interest.
- Indirect Immunohistochemistry: uses an unlabeled primary antibody that is detected with labeled secondary antibodies, amplifying the signal and providing greater technical flexibility.
Microscopic Detection of Gene Sequences
- In situ Hybridization (ISH): a technique that uses labeled cDNA probes to detect specific gene sequences or mRNAs in cells microscopically.
Labels Used in Immunohistochemistry
- Fluorescent compounds: used for light microscopy.
- Peroxidase: used for light microscopy.
- Gold particles: used for Transmission Electron Microscopy (TEM).
Tissue Section Interpretation
- Minor artifacts, such as spaces and precipitates, can be introduced during tissue processing, slide preparation, and staining.
- These artifacts are not present in living tissue and must be recognized to avoid misinterpretation.
- Tissue sections are essentially 2D representations of 3D structures.
- It is crucial to understand this 2D-3D relationship to correctly interpret and study tissue sections.
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Description
This quiz covers the process of preparing tissue samples for study, including fixation, dehydration, embedding, and sectioning. It explains the role of chemical fixatives and solvents in preserving tissue structure and preparing it for microscopy.
