Histology Study Guide: Focus on Cell Staining and Tissue Preparation Techniques

Histology

study of tissues

What is the goal of histology

To learn microanatomy of cells, tissues and organs to correlate structure and function

Physiology

The study of normal body function based on the integration of structure and function

Cells

Basic structural and function units of tissues that make up organ systems

Ways of studying tissue

Microscopy and appearance of the tissue

Microscopy

Aids in visualizing the structural features and arrangement of the cells and tissues

Appearance of the Tissue

Useful in distinguishing between normal and abnormal tissue and diagnosing disease

Tissues

Distinctly organized aggregation of cells (& their products aka ECM) that are structurally related, perform specific and interrelated functions, and have the ability to communicate

4 basic types of tissues

epithelium, connective, muscle, nervous

Morphology

shape

Epithelium basic characteristics

1 or more layers of cells connected to each other that cover body surfaces, lines body cavities, tubes, ducts, and forms glands

Epithelium basic function

Protect the internal environment of body and regulates the exchange of materials between the internal and external environment. Morphology and number of layers determines function

Epithelium specific functions

protective barrier, absorption, secretion and transport/exchange

Connective Tissue basic characteristics

Composed of cells and extracellular fibers embedded in a protein rich gel-like matrix (aka ECM)

CT basic function

Underlies and supports, connects and protects the other basic tissues structurally and functionally

Types of CT

CT proper (loose & dense), Cartilage, bone, blood, and adipose

Types of CT Proper

Loose (flexibility and support) and dense (flexibility and strength)

Muscle Tissue basic characteristics

Contractile cells responsible for movement

Muscle Tissues composition

composed of intracellular contractile proteins (actin and myosin)

Types of Muscle Tissues

Skeletal, Smooth, and Cardiac

Skeletal Muscles

Attach to bone

Smooth muscles

Form walls of hollow organs and blood vessels

Cardiac muscles

Compose the heart

Nervous Tissue composition

Neurons and supporting neuroglial cells

Nervous Tissue basic function

transmit, receive, process, integrate and conduct electrical impulses that carry information

Workflow of Studying Tissue

Fixing, Embedding, Cutting, Staining, and Microscopy

Fixation goal

preserve tissue structure and take a "snapshot" of tissue by hardening tissue and cross linking the amino acids in proteins

Fixation functions

Termination of cell metabolism

Prevent Enzymatic Degradation

Kill Pathogens

Hardens Tissue

Preserves molecular composition

Fixation Cons

Does not react well with lipids, must use Osmium tetoxide to fix lipids

Embedding goal

Embed tissue in a solid media to facilitate sectioning

Embedding Steps

Dehydration via alcohol in steps

clearing solution removes alcohol and causes transparent tissue

Placed in melted paraffin

Paraffin cools

Sectioning goal

prepare tissue for staining

Sectioning instrument

Microtome

Planes of Sectioning

Cross Section, Longitudinal section

Oblique Section

Staining Goal

permit examination of tissue

2 Staining Methods

Histochemical Staining and Differential Staining

Histochemical Staining

Dyes react with specific biological compounds demonstrating specific location through the use of dyes or fluorescent dye-labeled antibodies that with react with specific biological components

Differential Staining

Aka counterstaining

Most common usage of multiple dyes

Uses both acidic and basic dyes

Stain reflects the cell's boundaries and structural and functional features of a cell

Provides contrast

H&E most common dyes

H&E

Hematoxylin (basic, blue) and Eosin (acidic and pink)

Basic Dyes

Positively charged and react with negatively charged components (RNA, DNA) such Hematoxylin

Acidic Dyes

Negatively charged and react with positively charged components (plasma membrane and extracellular proteins associated with cytoplasmic organelles) such as Eosin

Things that do not stain well with H&E

Lysosomes, Peroxisomes, sER, Golgi, lipids, and euchromatin

Basophilic

Base loving (turns blue with hematoxylin)

Acidophilic

Acid loving (turns pink with Eosin)

Perioidc acid-Schiff (PAS)

Stains complex carbohydrates- including polysaccharides, glycoproteins, mucin, complex

Magenta colored stain

Wright-Giemsa stain

Red and White blood cells

RBC are red/orange and WBC nuclei are blue

Mallory-trichrome stain

connective tissue, collagen turns blue, nuclei are red, and cytoplasm is pink

Silver (elastin) stain

elasitc fibers, reticular fibers, and nervous tissue that is black/purple

Alternative Tissue Preparation Methods

Whole-mount, smears, teased preparations, Dry Ground Preparations, and Decalcification preparation

Whole-mounts

entire section is placed on slide for microscopic examination (small tissue samples like retina)

Smears

Sample of material is spread thinly on a slide for microscopic examination of individual cells

Cytology

Study of cells (uses smears)

Teased Preparations

Small area of tissue is teased apart and stained to show morphology (CT, muscle and nerve fibers)

Dry Ground preparation

Calcified tissue studied by grind down dried samples that has not been decalcified and then sectioned (organic material and cells removed)

Decalcification preparation

Calcified tissues studied by being decalcified prior to embedding and using regular H&E staining (permits study of cells and organic matrix by removing calcified material)

Artifacts in tissue processing

A structure not normally present and occurring likely due to an error in processing

Examples of artifacts

Adipose tissue appearing vacuolated (alcohol removes lipids during fixation), shrinkage (alcohol too fast during fixation), and wrinkles or folds (improper mounting)