Preparazioni Istologiche e Analisi

Histological Preparations

• Histological preparations are processed tissue samples used in microscopy.
• They are crucial for visualization and analysis of cellular structures and tissue organization.
• Preparation involves several steps to preserve and highlight specific features.

Sample Collection and Fixation

• Tissue samples are taken from living organisms or post-mortem specimens.
• Fixation is the initial step preserving tissue structure and preventing autolysis.
• Fixatives: Formaldehyde (most common), glutaraldehyde, alcohol.

Dehydration

• Removing water from fixed tissue is crucial for embedding.
• A graded series of alcohol solutions (increasing concentration) is used.
• This ensures tissue maintains form and structure.

Clearing

• After dehydration, a clearing agent replaces alcohol.
• Xylol or benzene allows infiltration of the paraffin embedding medium.
• This step promotes tissue compatibility and hardening.

Embedding

• Paraffin wax is a common embedding medium.
• Tissue is immersed in melted wax for complete infiltration.
• This creates solid blocks for sectioning.

Sectioning

• The embedded tissue block is sliced into thin sections.
• Microtone is used for precise and uniform sectioning.
• Section thickness varies based on objectives and techniques.

Staining

• Staining visualizes specific structures.
• Different stains target different components.
• Examples: H&E (Hematoxylin and Eosin), Periodic Acid-Schiff (PAS).

Mounting

• Sections are mounted on glass slides.
• Mounting medium (e.g., glycerin jelly, synthetic agents) is used.
• This ensures section adhesion to the slides for viewing.

Imaging of Histological Preparations

• Microscopy techniques visualize histological preparations.
• Light microscopy (LM) and electron microscopy (EM) are primary methods.
• Light microscopy uses visible light for magnification and visualization.

Light Microscopy (LM)

• Different staining techniques highlight components (nucleus—blue with hematoxylin; cytoplasm—pink with eosin).
• LM allows rapid tissue sample examination.
• Types of light microscopes include bright field and fluorescence microscopes.

Electron Microscopy (EM)

• Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are types of EM.
• TEM provides high-resolution internal tissue structure images.
• SEM provides high-resolution surface feature images.

Image Analysis and Interpretation

• Visualization is a first step; analysis is crucial.
• Histological images are analyzed for cellular and tissue organization.
• Software packages analyze images (e.g., quantifying cell densities, measuring tissue thickness).

Specialized Preparation Methods

• Immunohistochemistry uses labeled antibodies to visualize specific proteins.
• Immunofluorescence uses fluorescent dyes to visualize proteins.
• Other techniques include in situ hybridization.