Histology

Is the study of microscopic anatomy (microanatomy) of cells and tissues of plants and animals.
It is commonly performed by examining cells and tissues under a light microscope or electron
microscope, the specimen having been sectioned (cut into a thin cross section with
a microtome), stained, and mounted on a microscope slide. Histological studies may be
conducted using tissue culture, where live human or animal cells are isolated and maintained in
an artificial environment for various research projects. The ability to visualize or differentially
identify microscopic structures is frequently enhanced through the use of histological stains.
Histology is an essential tool of biology and medicine.

-- Four Major Types of Animal Tissue--

1. Epithelial

Epithelial tissue is made of closely-packed cells arranged in flat sheets. Epithelia form the
surface of the skin, line the various cavities and tubes of the body, and cover the internal
organs.

Subsets of Epithelia

Epithelia that form the interface between the internal and external environments.
o Skin as well as the lining of the mouth and nasal cavity. These are derived
from ectoderm.
o Inner lining of the GI tract, lungs, urinary bladder, exocrine glands, vagina and
more. These are derived from endoderm.

The apical surface of these epithelial cells is exposed to the "external environment",
the lumen of the organ or the air. [View example]

Mesothelia. These are derived from mesoderm.

o pleura the outer covering of the lungs and the inner lining of the thoracic
(chest) cavity.
o peritoneum the outer covering of all the abdominal organs and the inner lining
of the abdominal cavity.
o pericardium the outer lining of the heart.
Endothelia. The inner lining of the heart, all blood and lymphatic vessels derived
from mesoderm.

The basolateral surface of all epithelia is exposed to the internal environment (ECF). The
entire sheet of epithelial cells is attached to a layer of extracellular matrix that is called the
basement membrane or, better (because it is not a membrane in the biological sense),
the basal lamina.

The function of epithelia always reflects the fact that they are boundaries between masses of
cells and a cavity or space. Some examples:
 The epithelium of the skin protects the underlying tissues from
o mechanical damage
o ultraviolet light
o dehydration
o invasion by bacteria
The columnar epithelium of the intestine
o secretes digestive enzymes into the intestine;
o absorbs the products of digestion from it.
An epithelium also lines our air passages and the alveoli of the lungs. It secretes mucus
which keeps it from drying out and traps inhaled dust particles. Most of its cells have cilia
on their apical surface that propel the mucus with its load of foreign matter back up to
the throat.

2. Muscle

Three kinds of muscle are found in vertebrates:

Skeletal muscle is made of long fibers whose contraction provides the force of
locomotion and other voluntary body movements.
Smooth muscle lines the walls of the hollow structures of the body, such as the
intestine, urinary bladder, uterus, and blood vessels. Its contraction, which
is involuntary, reduces the size of these hollow organs.
The heart is made of cardiac muscle.

3. Connective

The cells of connective tissue are embedded in a great amount of extracellular material.
This matrix is secreted by the cells. It consists of protein fibers embedded in an amorphous
mixture of protein-polysaccharide ("proteoglycan") molecules.

Supporting connective tissue

Gives strength, support, and protection to the soft parts of the body.

cartilage. Example: the outer ear

bone. The matrix of bone contains collagen fibers and mineral deposits. The most
abundant mineral is calcium phosphate, although magnesium, carbonate, and fluoride
ions are also present. [More on bone]

Dense connective tissue

Often called fibrous connective tissue.

Tendons connect muscle to bone. [View] The matrix is principally Type I collagen, and
the fibers are all oriented parallel to each other. Tendons are strong but not elastic.
Ligaments attach one bone to another. They contain both collagen and also the
protein elastin. Elastin permits ligaments to be stretched.
Loose connective tissue

It is distributed throughout the body. It serves as a packing and binding material for most of our
organs. Sheets of loose connective tissue that bind muscles and other structures together are
called fascia. Collagen, elastin, and other proteins are found in the matrix of loose connective
tissue.

Both dense and loose connective tissue are derived from cells called fibroblasts [View], which
secrete the extracellular matrix.

Adipose tissue

Adipose tissue is "fat". There are two kinds found in mammals:

white adipose tissue (WAT) in which the cells, called adipocytes, have become almost
filled with oil. The oil is confined within a single membrane-enclosed droplet. Virtually all
of the "fat" in adult humans is white adipose tissue.
brown adipose tissue (BAT) in which the adipocytes contain many small droplets of oil
as well as many mitochondria.

White adipose tissue and brown adipose tissue differ in function as well as cellular structure.
New adipocytes in white adipose tissue are formed throughout life from a pool of precursor
cells. These are needed to replace those that die (after an average life span of 10
years). Whether the total number of these adipocytes increases in humans becoming fatter as
adults is still uncertain. If not, why do so many of us get fatter as we age? Because of the
increased size of individual adipocytes as they become filled with oil.

The adipocytes of white adipose tissue secrete several hormones,

including leptin and adiponectin.

4. Nerve

Nerve tissue is composed of

nerve cells called neurons and

glial cells.

Neurons

Neurons are specialized for the conduction of nerve impulses. A typical neuron consists of

a cell body which contains the nucleus;

a number of short fibers dendrites extending from the cell body
a single long fiber, the axon.

The nerve impulse is conducted along the axon.

BASIC TECHNIQUES

Preparation of histological sections

1. Fixation

In order to preserve tissues and prevent structural change or breakdown of the components of
the tissues, it is necessary to stabilize or fix the tissue. The fixative needs to preserve the
tissues as close as possible to the living state. The fixatives commonly stabilize or denature
proteins. A widely used fixative is formaldehyde, which has the advantage of being cheap and
penetrates tissues rapidly. For better fixation, it is necessary use pH buffers in the fixative.

2. Embedding

The most commonly used embedding or support medium is paraffin wax, with a melting point of
about 56C. Prior to infiltration of the tissue with molten wax, it is necessary to remove all the
water from the tissue (dehydration). Dehydration is achieved using an ascending series of
alcohols (70%, 95%, 100%). This is followed by tissue immersion in a wax solvent such as
xylene or chloroform. The tissue is then transferred to molten paraffin wax (in an embedding
oven) for a couple of hours. The tissue is then placed in a square or rectangular mold, and
orientated in the required position, prior to adding hot wax to form a wax block.

3. Microtomy

Sections of the tissue embedded in the wax block are cut on a machine, known as a microtome,
using special knives (nowadays these are disposable). Typically series or ribbons of sections
are cut at a thickness of 6-8mm. The sections are transferred to the surface of a hot waterbath
(where the sections flatten and lose any wrinkles). Sections are collected on glass microscope
slides (standard dimensions of 3 x 1 inches). In order for the sections to adhere to the slides
they are dried for up to 24 hours in a drying oven (at a temperature of about 40C). This
prevents sections falling off the slides in the later stages of preparation.

4. Staining

The most common staining technique is known as Hematoxylin and Eosin (or H&E) staining. In
order to stain the sections the wax needs to be removed. This is done using a wax solvent such
as xylene. The slide is then hydrated using a series of descending alcohols (100%, 95%, 70%)
and then water. The slide is then immersed in Hematoxylin stain, rinsed in running water
(preferably alkaline), followed by staining with Eosin, and rinsing in water.

5. Permanent Mounting

After staining the sections are again dehydrated with ascending alcohols (95%, 100%) and
xylene, prior to covering with a mountant and a glass coverlip. Mountants need to have good
optical properties. The slide is left for at least 24 hours for the mountant to dry. The finished
(permanent) slide with its stained tissues can then be examined under the microscope.