Specimen Preparation Techniques in Histology
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Specimen Preparation Techniques in Histology

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Questions and Answers

Which dye stains nuclei blue by binding to negatively charged structures?

  • Haematoxylin (correct)
  • Oil Red O
  • Alcian Blue
  • Eosin

    • What color does the cytoplasm stain when using Eosin?

  • Black
  • Blue
  • Pink (correct)
  • Green

    • Which of the following dyes is used to stain fat in unfixed frozen sections?

  • DAPI
  • Masson’s Trichrome
  • Oil Red O (correct)
  • Haematoxylin

    • What is the color of collagen when stained with Millers Sirius Red?

    <p>Red/Pink</p> Signup and view all the answers

    Alcian Blue is primarily used to stain which type of cellular structure?

    <p>Mucin</p> Signup and view all the answers

    Which staining technique is often used to visualize connective tissue?

    <p>Masson’s Trichrome</p> Signup and view all the answers

    What characteristic feature does DAPI have in cell staining?

    <p>It binds specifically to A-T rich regions in DNA.</p> Signup and view all the answers

    In the Masson’s Trichrome staining, what color indicates the presence of nuclei?

    <p>Blue</p> Signup and view all the answers

    What are the four main types of tissue in the human body?

    <p>Muscle, Nervous, Epithelial, Connective</p> Signup and view all the answers

    What is the primary aim of specimen preparation in histology?

    <p>To embed a tissue in a solid medium that supports its structure</p> Signup and view all the answers

    Which method is NOT part of the stages of tissue processing?

    <p>Cryopreservation</p> Signup and view all the answers

    What role does the extracellular matrix (ECM) play in tissue structure?

    <p>It provides mechanical support and transports nutrients to cells</p> Signup and view all the answers

    What is a primary characteristic feature of histology as a science?

    <p>It is a descriptive science emphasizing observation</p> Signup and view all the answers

    Which of the following is NOT an application for histology techniques?

    <p>Assessing metabolic rates of tissues</p> Signup and view all the answers

    What is the main purpose of staining techniques in histology?

    <p>To provide contrast to visualize cellular and sub-cellular structures</p> Signup and view all the answers

    During dehydration in specimen preparation, which substance is primarily used to remove water?

    <p>Alcohols</p> Signup and view all the answers

    What is a primary advantage of confocal microscopy?

    <p>Excellent resolution in thick samples</p> Signup and view all the answers

    Which type of ELISA allows for the detection of both antibodies and antigens?

    <p>Sandwich</p> Signup and view all the answers

    What is a disadvantage of using confocal microscopy?

    <p>Increased sensitivity to noise</p> Signup and view all the answers

    What role does the enzyme play in the ELISA process?

    <p>It acts as a reporter through color change</p> Signup and view all the answers

    What is the purpose of a standard curve in ELISA?

    <p>To estimate the unknown concentration of a sample</p> Signup and view all the answers

    What is the main purpose of fixation in specimen preparation?

    <p>To preserve tissue structure and prevent degradation</p> Signup and view all the answers

    Which of the following is a characteristic of a good clearing agent?

    <p>Is completely miscible with the embedding medium and dehydrating fluid</p> Signup and view all the answers

    In the dehydration process, which series of ethanol concentrations is typically used?

    <p>10%, 20%, 50%, 70%, 95%</p> Signup and view all the answers

    Which of the following agents is considered a heavy metal fixative?

    <p>Osmium tetroxide</p> Signup and view all the answers

    What is the initial step in the dehydration process of tissue specimens?

    <p>Removing fixative and water</p> Signup and view all the answers

    Which fixative type cross-links amine groups of proteins?

    <p>Aldehyde</p> Signup and view all the answers

    Why is it important to include a choice of clearing agent based on tissue type?

    <p>To maintain the integrity of the tissue structure during clearing</p> Signup and view all the answers

    What is a key characteristic of the embedding process in specimen preparation?

    <p>It provides support during sectioning</p> Signup and view all the answers

    Which embedding medium is suitable for sections ranging from 100 to 300 μm?

    <p>Agar</p> Signup and view all the answers

    What is one property that paraffin wax is improved for histological purposes?

    <p>Decreases melting point</p> Signup and view all the answers

    What is essential to ensure about the paraffin wax before embedding tissue?

    <p>Must be rapidly cooled after embedding</p> Signup and view all the answers

    What is the optimal section thickness for microtome sectioning for light microscopy?

    <p>4 - 40 μm</p> Signup and view all the answers

    Which statement about cryo-embedding is correct?

    <p>Tissue samples are frozen in liquid nitrogen or oxygen.</p> Signup and view all the answers

    What is the purpose of staining in microscopy?

    <p>To allow for the examination of tissues by light microscopy</p> Signup and view all the answers

    What type of stain is Haematoxylin and Eosin (H&E)?

    <p>A charge-based, general purpose stain</p> Signup and view all the answers

    What is the correct sequence for clearing and staining tissues on slides?

    <p>Cleared in Xylene, rehydrated through alcohol series, washed in water</p> Signup and view all the answers

    Study Notes

    Specimen Preparation - Fixation

    • Purpose: To prevent tissue degradation and maintain structural integrity.
    • Fixation Methods:
      • Aldehydes: Commonly used, cross-link proteins.
      • Ketones: Example - acetone.
      • Alcoholic Fixatives: Example - methanol, coagulates proteins.
      • Zinc Fixatives: Reversible fixation.
      • Heavy Metals: Example - osmium tetroxide.
      • Freezing: Utilizes temperature to preserve tissue.

    Specimen Preparation - Dehydration

    • Purpose: Removing water from tissue to prepare it for embedding.
    • Process: Gradual dehydration using a series of increasing ethanol solutions (10% to 100%).

    Specimen Preparation - Clearing

    • Purpose: Replacing the dehydrating fluid with a fluid miscible with both the dehydrating agent and the embedding medium.
    • Considerations:
      • Tissue type
      • Processing method
      • Processing conditions (temperature, vacuum, pressure)
      • Safety factors
      • Cost and convenience
    • Common Clearing Agents:
      • Xylene
      • Toluene
      • Chloroform
      • Benzene
      • Petrol
      • Histo-clear®
      • HISTOCHOICE

    Specimen Preparation - Embedding

    • Purpose: Surrounding tissue with a solid medium to provide support during sectioning.
    • Embedding Mediums:
      • Paraffin Wax: Used for sections ≥ 3 μm.
      • Plastic Resin: Used for very thin sections (0.5 - 1 μm).
      • Polymerizing Resin: Examples - Epoxy, Aradite.
      • Cryo-embed Medium: Used for sections ≥ 5 - 100 μm.
      • Agar: Used for sections 100 - 300 μm (unfixed) and 10 - 20 μm (fixed).

    Specimen Preparation - Embedding - Paraffin Wax

    • Properties: Mixture of solid hydrocarbons, density about two-thirds that of dried protein, slightly more elastic.
    • Considerations:
      • Melting point range (39°C to 68°C).
      • Properties can be improved by adding substances to the wax:
        • Improve ribboning
        • Increase hardness
        • Decrease melting point
        • Improve adhesion between specimen and wax
      • Wax should be free of clearing agents and dust particles.
      • Rapid cooling after embedding reduces crystal size.

    Slide Preparation - Sectioning

    • Process: Using a microtome to cut thin sections (4 - 40 μm) from embedded tissues.
    • Mounting: Sections are typically mounted onto glass slides.

    Cryo-embedding & Sectioning

    • Process:
      • Tissue samples are rapidly frozen using liquid nitrogen (LN2) or carbon dioxide (CO2).
      • Embedded using a medium that is solid below -10°C.
      • Sections are cut at > 6 μm using a cryostat.
      • Slide Storage: Slides must be stored below -20°C.
      • Slides often fixed before staining using alcohol, chloroform, or a 1:1 mixture.

    Staining

    • Purpose: To visualize tissues and cells for light microscopy.
    • Process:
      • Slides are cleared in xylene.
      • Rehydrated through an alcohol series.
      • Washed in water to remove alcohol.
      • Stained.
      • Dehydrated.
      • Cleared in xylene.
      • Mounted using xylene medium and a glass coverslip.

    Staining - Haematoxylin and Eosin (H&E)

    • Principle: Charge-based staining.
      • Haematoxylin: Cationic (+) dye, binds to negatively charged (acidic) structures like the nucleus, resulting in blue staining.
      • Eosin: Anionic (-) dye, binds to basic structures in the cell (e.g., amine groups on proteins), resulting in pink staining of the cytoplasm.

    Staining - DAPI

    • Principle: Fluorescent stain that binds to A-T rich regions of DNA.
    • Application: Staining both live and fixed cells, nuclei appear blue under fluorescence microscopy.
    • Excitation/Emission:
      • Excitation wavelength: 350 nm
      • Emission wavelength: 470 nm

    Staining - Alcian Blue

    • Principle: Mucin stain that binds to specific types of mucin, resulting in blue staining.
    • Application: Staining cartilage, acid mucins, and proteoglycans.
    • Compatibility: Can be used with Hematoxylin and Eosin (H&E), nuclear fast red, and Van Gieson stains.

    Staining - Oil Red O

    • Principle: Used to stain fat in unfixed frozen sections.
    • Application: Visualizing fat droplets in frozen sections.
    • Note: Processing can remove fat content from cells and tissues.
    • Coloring:
      • Fat: Brilliant red
      • Nuclei: Blue

    Staining - Miller's Sirius Red

    • Principle: Combination stain viewed under Köhler illumination and phase contrast.
    • Köhler Illumination:
      • Elastin: Dark purple/Black
      • Collagen: Red/Pink
    • Phase Contrast:
      • Collagen fibers appear birefringent (double refracting).

    Staining - Masson's Trichrome

    • Principle: Commonly used to stain connective tissue.
    • Coloring:
      • Nuclei: Blue
      • Cytoplasm, muscle, erythrocytes, keratin: Bright red
      • Collagen: Green or blue (depends on the specific variant of the technique).

    Staining - PAS (Periodic Acid Schiff)

    • Principle: Basic fuchsin that reacts with aldehyde groups.
    • Application: Histology (study of tissues).

    Microscopy - Confocal

    • Principle: Uses a laser to excite fluorescent dye in tissue or cells one spot at a time.
    • Advantages:
      • Excellent resolution in thick samples.
      • Greater number of fluorophores can be used as specific wavelengths are utilized to illuminate samples.
      • Collects light from a single focal plane.
    • Disadvantages:
      • Photobleaching and phototoxicity.
      • Increased sensitivity to noise.
      • Technical method - labor intensive.

    Enzyme Linked Immunosorbent Assay (ELISA)

    • Principle: Uses antibodies to detect specific antigens or antibodies in a sample.
    • Types:
      • Sandwich
      • Indirect
      • Competitive
    • Advantages: Highly sensitive and specific.
    • Procedure:
      • Antigen or antibody is bound to a solid surface.
      • A specific antibody is added to detect the bound antigen or antibody.
      • An enzyme-linked antibody is then added.
      • A substrate for the enzyme is added, resulting in a color change.
      • The color intensity is directly proportional to the amount of antigen or antibody present in the sample.

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    Histology Techniques PDF

    Description

    Test your knowledge on the essential techniques of specimen preparation in histology, including fixation, dehydration, and clearing processes. Learn about various methods like aldehydes, alcohols, and the importance of each step in preserving tissue. This quiz covers critical aspects necessary for effective preparation and analysis of biological specimens.

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