HISTOLOGY I

Introduction

• Lecturers: Dr. Mustafa Ghanim & Dr. Fatina Hanbali
• Delivery date: 01/02/2021

VISUALIZING SPECIFIC MOLECULES

Methods of Visualization

• Specific macromolecules in tissue sections can be identified using tagged compounds or macromolecules.
    - Tagged compounds must bind specifically to the molecule of interest.
    - These compounds must be visible under light or electron microscopes, often through detectable labels.

Common Identification Techniques

• Flourescent compounds, radioactive atoms, enzymes, and metal particles are widely used labels.
    - Fluorescent Compounds: Allow visualization through fluorescence microscopy.
    - Radioactive Atoms: Detected through autoradiography.
    - Enzymes (e.g., peroxidase): Detected through histochemistry.
    - Metal Particles: Typically gold, visible with light and electron microscopy.
    - These techniques are used for detecting specific sugars, proteins, and nucleic acids.

Specific Molecules Example

• Phalloidin: Extracted from the mushroom Amanita phalloides, interacts with actin protein in microfilaments.
• Protein A: Purified from Staphylococcus aureus; binds to the Fc region of antibodies for localization purposes.
• Lectins: Glycoproteins from plant seeds that bind carbohydrates; facilitate staining of specific glycoproteins.

AUTORADIOGRAPHY

Overview

• A method for localizing newly synthesized macromolecules in cells or tissue sections using radioactively labeled metabolites (nucleotides, amino acids, sugars).
    - These metabolites are incorporated into macromolecules and emit weak radiation.

Methodology

1. Tissue sections are coated with photographic emulsion in a darkroom.
2. Silver bromide crystals act as microdetectors for radiation, similar to light in photographic film.
3. After adequate lightproof exposure, the slides are developed photographically.
     - Radiation reduces silver bromide into metallic silver, visible as black grains.

Applications

• Allows histological analysis of replicating cells using radioactive precursors like tritium-labeled thymidine.
• Mapping protein production and migration in cells can be observed through sequential autoradiography.

ENZYME HISTOCHEMISTRY

Definition

• Enzyme histochemistry is a technique used to localize specific enzymatic activities within cellular structures.

Principles and Process

• Using unfixed or mildly fixed tissues to preserve endogenous enzymes.
    - Sections are prepared using cryostats to avoid enzyme degradation.
• Procedure:
    1. Tissue sections are immersed in the enzyme substrate solution.
    2. Enzyme action is allowed to proceed on the substrate.
    3. The section is then treated with a marker compound detectable by microscopy.
    4. The final product is insoluble, precipitating at the enzyme's location.

Examples of Enzymatic Reactions

• Phosphatases: Remove phosphate groups from macromolecules.
• Dehydrogenases: Transfer hydrogen ions in reactions, relevant to enzymes in the citric acid cycle.
• Peroxidase: Oxidizes substrates, facilitating histochemical identification.

IMMUNOHISTOCHEMISTRY

Concept

• Antigens and antibodies interact specifically, making them suitable for localization of proteins in tissues.
• Labeled antibodies aid in the identification of proteins, beyond enzymatic activity determined in enzyme histochemistry.

Antibody Characteristics

• Antibodies are glycoproteins from the immunoglobulin family, secreted by lymphocytes.
• They specifically bind to their antigens, recognized as foreign by the immune system.

Production of Antibodies

1. To produce antibodies against a target protein, the isolated protein is injected into a different animal species (e.g., human protein into a rabbit).
2. The animal recognizes the protein as foreign, producing antibodies specific to it.
3. Polyclonal antibodies are then collected from the animal's plasma, each recognizing different parts of the protein.

Monoclonal Antibodies

• Hybridoma cells can be created by fusing lymphocytes with tumor cells; this allows indefinite culture of monoclonal antibodies targeted at specific antigens.
• Monoclonal antibodies have advantages in specificity and less cross-reactivity compared to polyclonal antibodies.

Application in Tissue Studies

• Tissue sections are incubated with either monoclonal or polyclonal antibodies, binding to target proteins for visualization.
• Visualization methods:
    - Fluorescent tagging, enzyme labeling, or electron-dense particles.

Indirect vs Direct Methods

• Direct Method: Involves labeled antibodies directly binding to the protein.
• Indirect Method: Uses a secondary labeled antibody that enhances signal sensitivity through additional binding steps, widely used in research and diagnostics.
  

Biotin-Avidin Technique

• Other amplification methods exist, such as using biotin-avidin techniques to enhance detection signals.

HYBRIDIZATION TECHNIQUES

Basics of Hybridization

• Refers to specific binding between complementary nucleotide sequences of DNA or RNA.

Key Applications

1. Determining presence of specific DNA sequences, such as genes.
2. Identifying cells with active transcription of specific mRNAs.
3. Localizing specific genes on chromosomes.

Process

• Initial denaturation of nucleic acids, followed by annealing of labeled probes to complementary sequences.

Probes

• Probes can be obtained via cloning, PCR amplification, or chemical synthesis and are tagged for visibility.

Final Steps

• Finalized by washing off unbound probes and visualizing the hybridization results through their labels.

MEDICAL APPLICATIONS

• Immunohistochemistry is vital for diagnosing various diseases, including cancers and viral infections, making it crucial for personalized medicine.

FIGURES

• Figure 1-9 shows microscopic autoradiography.
• Figure 1-10 displays enzyme histochemistry examples.
• Figure 1-11 illustrates immunocytochemistry techniques.
• Figure 1-12 depicts cells stained by immunohistochemistry.
• Figure 1-13 demonstrates in situ hybridization.

TABLE

• Table 1-1 lists antigen examples related to specific diseases, showcasing practical applications of immunohistochemistry in diagnostics.