Histology: Tissues and Microscopy

Histology Overview

• Histology is microscopic anatomy and the study of tissues, cells, and the extracellular matrix (ECM).
• Histology studies how cells and tissues are arranged to constitute organs.
• The main tissue types are epithelium, connective, muscular, and nervous tissue.
• Bone and cartilage are special types of connective tissue.

Level of Organization

• The levels of organization are: chemical, cellular, tissue, organ, system, and organismal.
• Many tissue types are arranged in a way to perform a specific function within an organ.

Tissue Processing for Light Microscopy

• The process includes fixing, processing, embedding, cutting, microscopy, and staining.

Tissue Preparation for Light Microscopy

• To test a drug on a kidney, the drug is first tested on cells, then on an animal.
• Fixation involves placing tissue in chemical solutions to cross-link proteins, inactivate enzymes, and preserve cell and tissue structure.
• Fixation prevents tissue digestion by enzymes (autolysis) or bacteria.
• Dehydration transfers tissue through increasing alcohol solutions to remove all water

Clearing

• Alcohol is removed using organic solvents miscible with both alcohol and paraffin (wax).
• Wax replaces water to prepare the tissue for further processing.

Infiltration and Embedding

• Infiltration involves placing tissue in melted paraffin until completely infiltrated.
• Embedding involves placing paraffin-infiltrated tissue in a mold with melted paraffin and allowing it to harden.
• Trimming involves trimming the resulting paraffin block to expose the tissue for sectioning (slicing) on a microtome.
• A trimmed sample can be stored for hundreds of years if not exposed to chemical change.

Sectioning

• Sections must be thin (7-9 μm) for light to pass through for light microscopy.

Staining

• Staining enhances visualization and identification of cellular components for the LMS (light microscopy system).
• Separate specimens should not be mixed.
• Histopathology involves using histology to conduct pathological analysis of tissues.

Staining Principles

• Cells and extracellular materials are naturally colorless.
• Dyes form electrostatic linkages with ionizable radicals of macromolecules in tissues.
• Nucleic acids which have a negative anionic charge, attract basic dyes and are termed basophilic.
• Cationic components like proteins with ionized amino groups, stain readily with acidic dyes and are termed acidophilic.

Common Dyes

• Basic dyes: toluidine blue, alcian blue, and methylene blue Hematoxylin behaves like a basic dye and stains basophilic tissue components.
• DNA, RNA, and glycosaminoglycans ionize and react with basic dyes because of their acidic composition.
• Acid dyes: eosin, orange g, and acid fuchsin which stain mitochondria, secretory granules, and collagen.

Hematoxylin and Eosin (H&E)

• Routine stain because most cells and tissue are naturally colorless.
• Binds to ionizable molecules in cells based on charge.
• The nucleus appears bluish-purple because DNA and RNA attract the basic dye hematoxylin.
• The cytoplasm appears pink because many cytoplasmic components attract the acidic dye eosin.
• Regions with negatively charged molecules stain more basophilic.
• Cell nuclei consistently show basophilic staining due to nucleic acid content (e.g., pancreas tissue).

Special Stains

• Trichrome stains provide greater distinction among extracellular tissue components when studying connective tissue.
• The periodic acid-Schiff (PAS) reaction stains carbohydrate-rich tissue structures purple or magenta.
• Sudan black stains lipids, avoiding processing steps that remove lipids.
• Metal impregnation uses silver salts to visualize certain ECM fibers and specific cellular elements in nervous tissue to visualize reticular fibers.
• Immunostaining includes immunofluorescence and immunohistochemistry and uses antigen-antibody specificity.
• Toluidine Blue stains nerve tissue sections and highlights myelinated parts of the nerve that cannot be clearly seen with H&E.
• Alcian Blue identifies mucin (mucus present in glands and epithelial tissues) that H&E doesn't highlight.

Common Histological Stains and Their Colors

• Hematoxylin and eosin (H&E): stains the nucleus blue and the cytoplasm pink.
• Van Gieson method: stains collagen pink and muscle yellow.
• Trichrome method: stains connective tissue blue, cytoplasm pink, and nuclei dark brown.