Histology and Microscopy Techniques

• Histology involves tissue preparation and microscopy.

Tissue Processing Steps

• Fixation preserves tissue structure using formalin.
• Dehydration removes water using ethanol.
• Clearing uses xylene.
• Infiltration uses xylene/paraffin.
• Embedding uses paraffin.
• Sectioning involves cutting tissue into thin sections using a microtome.
• Mounting places sections on slides.
• Rehydration rehydrates the tissue.
• Staining applies stains for visualization.

Tissue Section Types

• Frozen sections use a cryostat and are a fast process, preserving lipids and antigens, often used during surgeries for rapid diagnosis.
• Semi-Thin/Ultra-Thin Slices involve tissue embedded with epoxy, plastic, or acrylic resin.
• Paraffin sections are good for long-term storage and are easy to section, but result in loss of lipids and some antigens.
• EM (Plastic Embedded - epoxy/acrylic) are difficult and time-consuming, allowing viewing of ultrastructures, and requires very thin sections.

Staining Techniques - Dye Types

• Basic Dyes examples are Hematoxylin, toulidine blue, methylene blue, staining acidic (basophilic) substances (e.g., RNA/DNA) that result in dark, blue/purple stain.
• Acidic Dyes examples are Eosin, acid fuchsin, staining basic (acidophilic) tissue (e.g., proteins) which result in pink stain.
• Lipid-Soluble Dyes examples are Sudan black/Oil Red, staining fats and oils.
• Multicomponent Dyes examples are PAS and Trichrome (Masson).
• PAS stains complex carbohydrates (glycogen) with an intense magenta.
• Trichrome stains collagen (blue), nuclei (black), and cytoplasm (pink).

Common Stains

• H/E Stain is the most common stain.
• Oil Red O / Sudan Black stains for lipids.
• PAS stains for complex carbohydrates.

Microscopy Types

• Light Microscopy

Bright Field

• Uses a halogen bulb, objective lenses (4x, 10x, 40x, 100x), and ocular lenses (10x).

Phase-Contrast

• Provides rapid and easy visualization and is for living, unstained cells, common in tissue culture labs.

Differential Interference

• Produces 3D image of living, unstained cells.

Confocal Laser Scanning

• Produces very sharp, clear image and it is used in biomedical sciences.

Fluorescence

• Produces brilliant colors with a dark background using customized stains to target certain cell macromolecules.

Electron Microscopy

• Transmission Electron (TEM) produces cross-sections of cell structures and shows very small organelle structures, helpful with membrane structures & organelles.
• Scanning Electron (SEM) shows detailed 3D surface features.

Histochemistry & Immunohistochemistry

• Histochemistry involves the treatment of tissue/cells for identification or specific chemical components or molecules; staining method relies on chemical reactions that tag specific enzymes or molecules in cells by producing a color change (e.g., Tags for phosphatase, peroxidase (Iron stain via Prussian Blue reaction)).
• Immunohistochemistry is a method for tagging specific proteins by utilizing interactions between labeled antibodies with their antigens present with antibody typically tagged with a fluorescent compound.

Four Basic Tissue Types

• Nervous tissue has Intertwining, elongated processes, no stroma, transmits nerve impulses, and is derived from the ectoderm.
• Epithelium tissue has Aggregated polyhedral cells, a small amount of stroma, lines body surfaces/cavities and glandular secretions, and is derived from the ecto, meso, and endo derms.
• Muscle tissue has Elongated contractile cells, a moderate amount of stroma, facilitates movement, and is derived from the mesoderm.
• Connective tissue has Several types of fixed and wandering cells, abundant amount of stroma, provides support and protection, and is derived from mesoderm.

Epithelium Functions

• Lines body surfaces/cavities
• Secretion (e.g., glands)
• Absorption (e.g., intestines)
• Contractility (e.g., mammary glands)

Epithelium Characteristics

• Polarity with Apical side facing cavity (may have microvilli and cilia), Lateral side contacting adjacent cells (has cell junctions), and Basal side between cell and connective tissue (basement membrane).
• Little extracellular space between cells.
• Shape/layer classifications: stratified, simple, columnar, squamous

Basal Lamina and Basement Membrane

• Basement membrane is a semi-permeable barrier, provides structure and polarity to epithelial cells, helps with filtering capillaries, migration, etc., and stains best with PAS because it has GAGs.
• Basal lamina is the upper layer of the basement membrane.

Cell Junctions (Lateral)

• Tight Junction (Zonula occludens) is located most apically, has Claudin junctional protein and actin cytoskeletal component.
• Adherens Junction (Zonula adherens) is located laterally, has Cadherin junctional protein and catenin with actin cytoskeletal components.
• Desmosomes (Macula adherens) are located at spot welds, have Desmogleins & desmocollins junctional proteins and intermediate filaments (keratin) cytoskeletal component.
• Gap Junctions (Communicating junctions) are located between neighboring cells, have Connexin (forms connexons) junctional proteins and none cytoskeletal component.
• Hemidesmosomes are located basally, have Attachment plaques containing integrins as junctional proteins and intermediate filament (keratin) as the cytoskeletal component.
• Tight Junctions define cell polarity and control the passage of substances between adjacent cells, with a distribution like a ribbon internally bracing the cells.
• Hemidesmosomes link the basal domain of an epithelial cell to the basal lamina.
• Gap Junctions are formed by channel-like structures that enable the passage of small molecules (~1.2 kd) between cells, connecting functionally two adjacent cells.

Apical Specializations

• Microvilli has actin filaments (organized) as its structure, is short in length are located in the intestines and have glycocalyx + microvilli = brush border.
• Stereocilia has actin filaments (not organized) as its structure, are longer, larger, and more sparse.
• Cilia has a 9+2 formation of microtubules and dynein structure, longer than microvilli in length, located in the respiratory tract and oviducts, ear hair cells with a basal body.
• Microvilli vs Cilia indicates Celiac's disease.

Epithelium II - Epithelium Classification & Function

• Simple squamous has Thin, flat cells with thin, flat nuclei, facilitates rapid exchange/diffusion of substances, and lines blood vessels/endothelium, air sacs/alveoli, and body cavities/mesothelium.
• Simple cuboidal has square/cube shaped cells, and round nuclei, facilitates absorption & secretion, and is found in the collecting tubule of kidneys.
• Simple columnar has tall, rectangle shaped cells, nuclei may be round or oval in a single row, facilitates absorption and often have microvilli/cilia and lines the intestinal mucosa.
• Stratified provides extra protection.
• Stratified squamous keratinized has dry cells, lose nuclei, and prevents loss of moisture.
• Stratified squamous nonkeratinized is wet and lines the esophagus and mucous membranes.
• Stratified cuboidal is uncommon, represents a double layer of cuboidal mostly in big glands.
• Stratified columnar is uncommon, found in the male urethra and inner eyelid.
• Transitional epithelium has dome shaped / umbrella cells can stretch/distend and is found in the bladder, ureters, and prostate; plaques at apical surface protect from hypertonic urine called urothelium.
• Pseudostratified columnar epithelium appears layered because of crowded arrangement of cells (no real organization of nuclei, usually ciliated), is actually simple layer of cells because each cell contacts basement membrane and is associated with goblet cells in respiratory tract and trachea, commonly called respiratory epithelium.
• Glands that are Endocrine release contents directly into blood and have no ducts.
• Glands that are Exocrine release contents to surface, have ducts (conduction), acinus (secretory), epithelial cells.
• Simple exocrine glands have main duct unbranched, can have multiple acinus.
• Compound exocrine glands have main duct branched.

Exocrine Gland Secretion

• Merocrine secretion occurs via exocytosis of secretory granules (e.g., salivary gland (serous or mucous)).
• Apocrine secretion occurs via loss of apical portion of cell, usually with lipid droplets (e.g., mammary gland).
• Holocrine secretion occurs via total disintegration of cell (e.g., sebaceous gland on hair and on skin).

Serous vs. Mucous Epithelial Cells

• Serous gland has liquid or watery secretion, apical side is eosinophilic, and basal side is basophilic due to displacement of nuclei and RER.
• Mucous gland has thick, viscous secretion of glycoproteins, larger than serous cells, and an apical region filled with pale staining mucin secretory granules and flattened nuclei at basal end.
• Mixed gland contains mucous cells (flat nuclei) + serous cells (round nuclei) = demilune

Simple Gland Types

• Simple Tubular: Intestinal glands
• Simple Coiled Tubular: Merocrine sweat glands
• Simple Branched Tubular: Gastric glands, Mucous glands of esophagus, tongue, duodenum
• Simple Alveolar (Acinar): Not found in adult; a stage in development of simple branched glands
• Simple Branched Alveolar: Sebaceous (oil) glands

Compound Gland Types

• Compound Tubular: Mucous glands (in mouth), Bulbourethral glands (in male reproductive system), Testes (seminiferous tubules)
• Compound Alveolar (Acinar): Mammary glands, Glands of respiratory passages
• Compound Tubuloalveolar: Salivary glands, Pancreas

Epithelial Cell Type Locations

• Transitional cells are found in Bladder, Ureters, Urethra, and Prostate.
• Pseudostratified cells are found in the Respiratory tract.
• Stratified Squamous cells are found in the Skin (epidermis), Mucous membranes (oral mucosa), Esophagus, and Cornea.
• Simple Columnar cells are found in the Intestine (epithelium).
• Stratified Columnar cells are found in the Male urethra, and Palpebral conjunctiva (inner eyelid).
• Simple Squamous cells are found in Blood vessels (endothelium), Alveoli, and Body cavities (mesothelium).
• Simple Cuboidal cells are found in Thyroid, Glandular ducts, Renal tubules, and Lens of the eye.