histology fundamentals for medical students

Histology,[help 1] also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope.[5][6] Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue.

There are four basic types of animal tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissues

All animal tissues are considered to be subtypes of these four principal tissue types (for example, blood is classified as connective tissue, since the blood cells are suspended in an extracellular matrix, the plasma)

• Epithelium
  • Simple epithelium
    • Simple squamous epithelium
    • Simple cuboidal epithelium
    • Simple columnar epithelium
  • Pseudostratified columnar epithelium
  • Stratified epithelium
    • Stratified squamous epithelium
    • Stratified cuboidal epithelium
    • Stratified columnar epithelium
    • Transitional epithelium
  • Multicellular glands
• Muscle tissue
  • Smooth muscle
  • Skeletal muscle
  • Cardiac muscle
• Connective tissue
  • General connective tissue
    • Loose connective tissue
    • Dense connective tissue
  • Special connective tissue
    • Cartilage
    • Bone
    • Hemopoietic
    • Blood
    • Lymph
• Nervous tissue
  • Central nervous system
  • Peripheral nervous system
  • Special receptors

Four basic types of human tissue can be stained and viewed using various histological techniques. Epithelium, connective tissue, muscle tissue, and nervous tissue have commonalities but look very distinct structurally after staining. Each stain exists to highlight an important feature or component within a tissue type. For example, one of the most common stains, Hematoxylin, is a basic dye that stains proteins a blue color, while Eosin stains proteins a pink color. These two stains are commonly used together to define intracellular organelles and proteins. Because of the variety of the proteins that exist, some stains were created to highlight a particular protein, which this review will discuss in the following sections. The benefit of using a special stain is that it can highlight the specific protein very well. However, because of its specificity, the other structures will not be seen. For this reason, multiple slides will often be created from a given specimen so that multiple stains can be performed to gather the full range of needed information.

Almost all tissue stains are performed on tissue that has been removed from the body. However, in rare instances, very specialize stains called vital stains can work on tissue remaining in the body. These stains are used for the identification of specific types of tissue and identification of abnormal tissue, so a subsequent biopsy can be more accurate in obtaining abnormal tissue

Tissue Preparation

Before specific staining can occur, tissue samples must undergo preparation through the following stages: Fixation, processing, embedding, sectioning, and sometimes antigen retrieval. In modern histology laboratories, most of these steps are automated.

.Fixation: Fixation uses chemicals to preserve the structure of the tissue in its natural form and protects it from degradation by irreversibly cross-linking proteins. Although several specialized fixatives are available, Neutral Buffered Formalin is a common choice for this step. The fixation step is vital to the rest of the histologic staining procedure because by retaining the chemical composition of the tissue, the sample is hardened and makes the sectioning phase easier. Paraffin-formalin is another effective fixative. Its benefit is that it is the fixative of choice for immunostaining; however, it requires preparation at the time of the fixation. Bouin is a fixative used for examining embryo and brain tissue because of its superior preservation of delicate nuclei and glycogen. Its downside is that it does not preserve kidney tissues well and also distorts mitochondrial structure.[1]

Dehydration: The addition of ethanol accomplishes the dehydration of a sample. It removed water from the sample and further hardens the tissue for eventual light microscopy. After ethanol is applied, and following the completion of tissue dehydration, xylene is used to remove the ethanol.[1]

Embedding: Embedding is the process of putting the sample into a paraffin wax or a plastic resin to enhance the process of extracting cellular structures. This step is to be performed with caution if the goal is to perform immunostaining because the paraffin wax will inhibit the penetration of antibodies, and lead to a false result.[1]

Sectioning: Sectioning involves mounting the specimen on a microtome and cutting it into sections. The preferred thickness is 4-5 micrometers so that it can be stained and put on a microscope slide for examination.[1]

Antigen Retrieval: This step is to retrieve antigens that could have been covered in the fixation and embedding stages. If the cross-linking of proteins conceals the antigen sites, there may not be as robust of an immunohistochemical response. Antigen retrieval is achieved through heating and proteolytic methods to break down the cross-links and reveal the epitopes and antigens that were previously covered.[1] Although this step carries the risk of denaturing both the fixative and the antigens themselves, a successful antigen retrieval method can lead to a much more effective immunostaining intensity.