Histology: Extracellular Matrix & Tissue Prep

• Histology involves studying the tissues of the body and their arrangement to form organs, also known as microscopic anatomy.
• Tissues have two main interacting components: cells and the extracellular matrix (ECM).

Extracellular Matrix (ECM)

• The ECM is organized and forms complex structures surrounding cells, such as collagen fibrils and the basement membrane.
• The ECM provides mechanical support to cells.
• The ECM transports nutrients.
• The ECM carries away metabolites and secretory products.

Tissue Preparation

• Ideal microscopic preparation preserves tissue so it retains structural features it had in the body.
• Tissue preparation steps include:
  • Numbering.
  • Fixation.
  • Dehydration.
  • Clearing.
  • Infiltration.
  • Embedding.
  • Trimming.
  • Sectioning.
  • Staining.
  • Mounting.

Fixation (Tissue Preparation)

• Fixation avoids tissue digestion via enzymes or bacteria.
• Fixation preserves structure and molecular composition.
• Fixation stops decomposition, putrefaction, and distortion after removal from the body.

Dehydration (Tissue Preparation)

• Dehydration involves removing water from tissue using ascending concentrations of alcohol.

Clearing (Tissue Preparation)

• Clearing removes the dehydrating agent from the tissues.
• Tissues become transparent during clearing.
• Xylene is used in the clearing process.

Infiltration (Tissue Preparation)

• Infiltration involves filling tissue gaps/cavities with a solid medium.
• Infiltration gives a firm consistency to the specimen.
• Infiltration removes the clearing agent.

Embedding (Tissue Preparation)

• Embedding involves placing impregnated tissue in a mold, containing medium, allowing it to solidify in a precisely arranged position.
• Embedding facilitates sectioning.
• Embedding stabilizes the tissue.

Sectioning (Tissue Preparation)

• Sectioning involves cutting tissue into uniformly thin slices.
• A microtome is used to slice specimens into thin sections.

Staining (Tissue Preparation)

• Staining involves applying dyes to tissue sections for studying patterns and physical characteristics of cells.
• Cells and tissues have an affinity for dyes.

Staining Classes

• Stains differentiate acidic and basic components of the cell.
• Stains differentiate fibrous components of the matrix.
• Metallic salts precipitate tissue, forming metal deposits.

Staining Dyes

• Basic dyes include Toluidine blue, Alcian blue, and Methylene blue.
• Acidic dyes include Eosin, Acid fuchsin, and Orange G.
• Acidophilic tissues include nucleic acid, glycosaminoglycans and acid glycoproteins.
• Orceins Weigart Elastic stain stains elastic fibers.
• Silver stain stains reticular fibers.
• Iron Hematoxylin stains striation of muscles, nuclei, and erythrocytes.
• Periodic Acid Schiff (PAS) stain stains glycogen and carbohydrate-rich molecules.
• Hematoxylin has an affinity for cartilage matrix.
• Hematoxylin provides powerful nuclear and chromatin staining capacity.
• Hematoxylin stains DNA of the nucleus.
• Eosin stains cytoplasmic components and collagen fibers and is for connective tissues.
• Trichromes (Mallory and Masson's) use 3 colors and distinguish extracellular tissue components better than H&E stain.
• Wright and Giemsa stain blood samples and stain eosinophil granules, red blood cells, basophil granules, nuclei of WBC, monocytes, lymphocytes, and cytoplasm.

Mounting (Tissue Preparation)

• Mounting protects the tissue specimen by using a mounting medium and coverslip.

Cells

• The main parts of a cell are the cytoplasm, nucleus, and plasma membrane.
• Cells and extracellular material comprise the tissues of multicellular animal organs.
• Cells are the basic structure and functional units of an organism.
• Eukaryotic and prokaryotic cells are two types of cells.

Plasma Membrane

• It's a microscopic membrane of lipids and proteins that forms the external boundary of cytoplasm/encloses a vacuole, regulating molecule passage.
• Endocytosis involves bulk uptake of material across the plasma membrane and folding/fusion of the membrane to form vesicles.
• Exocytosis involves membrane-limited cytoplasmic vesicle fusion with the plasma membrane which releases content into the extracellular space.
• Phagocytosis and Pinocytosis are a type of cell process.
• Apoptosis is the process of cell suicide/programmed cell death.

Chromosomes

• In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes.
• Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure.
• In humans, each cell contains 23 pairs of chromosomes, totaling 46.
• Autosomes are twenty-two of these pairs and look the same in both males and females.
• Sex chromosomes are the 23rd pair and differ between the males and females.

Cell Division

• Mitosis is somatic cell division, parent cell divides with identical chromosomal set.
• Meiosis is reproductive cell division involving specialized process with 2 divisions in cells that form sperm and egg cells.
• Cells produced are haploid with one chromosome from each pair in the body's somatic cell.

Cell Cycle

• The cell cycle consists of the period from the beginning of one division to the next
• The time it takes to complete one cell cycle is the generation time.

Phases of the Cell Cycle

• The Phases of the Cell Cycle are:
• Interphase - Longest Phase:
• G1 Phase - cell growth.
• S Phase - DNA replication.
• G2 Phase - prep for mitosis.
• M Phase (Mitotic Phase):
  • Prophase.
  • Metaphase.
  • Anaphase.
  • Telophase.
• Cytokinesis.

Interphase (Cell Cycle)

• The cell engages in metabolic activity for preparation for mitosis.
• Chromosomes are not discerned in the nucleus; dark spot called nucleolus may be visible.
• Cell contains a pair of centrioles (microtubule organizing centers in plants), organizational sites for microubules

Prophase (Cell Cycle)

• Chromatin in the nucleus condenses, becomes visible in the light microscope as chromosomes.
• Nucleolus disappears.
• Centrioles move to opposite ends of cell, some fibers cross, forming the mitotic spindle.

Prometaphase (Cell Cycle)

• Dissolution of the nuclear envelop & spindle fibers come in contact with the chromosomes

Metaphase (Cell Cycle)

• Chromosomes become arranged in the plane of the spindle equator, "equatorial or metaphase plate".

Anaphase (Cell Cycle)

• Splitting of the centromere marks this stage and binds chromatids duplicated on each chromosome.
• Mitotic spindle lengthens while astral microtubules shorten.
• Centrioles are pulled apart, chromatids of each duplicated chromosome drawn to opposite ends of the spindle.
• Achieves exact division of the duplicated genetic material.

Telophase (Cell Cycle)

• Chromosomes uncoil and regain their interphase conformation.
• Nuclear envelop and nucleoli become apparent.
• Plasma membrane indents the spindle equator and forms a circumferential furrow around the cell – the cleavage furrow which progresses until the cell is cleaved into two daughter cells.