Tissue processing steps

Fixation-
 Stabilizes tissue proteins to prevent further changes
such as decay.
 Most commonly achieved with 10% neutral buffered
formalin.
Dehydration
 Removes water from tissue because meled paraffin
wax is hydrophobic (immiscible with water) most of
water in the tissue must be removed before it can be
infiltrated by the paraffin.
 Alcohol is used.
 Gradual removal of water is preferred.
Clearing-
 Removes the dehydrant from the tissue in
preparation for infiltration
 Clearing is the process which leaves the tissue clear
and transparent.
Imperegnation (infiltration)-
  Displaces clearing agent in tissue with paraffin, in
preparation for paraffin embedding
Epithelial cell Junctions
Junctions- are specialized structures in the epithelia
that link (adhere) individual cells together to form a
functional unit.
Two main systems involved in cell adhesion:-
Cell membrane proteins function as specialized cell
adhesion molecules.
Specialized areas of cell membrane incorporated
into cell junctions.
Types of cell junctions:-
1. Tight junctions-
 Link cells to form impermeable barrier.
 These junctions act as barriers that prevent the
movement of molecules into the intercellular spaces.
 These junctions separate areas of cell membrane
that are specialized for absorption or secretion from
the rest of the cell membrane.
  In cells involved in active transport against a
concentration gradient, occluding junctions prevent
back diffusion of transported substances.

2. Anchoring junctions-
 Link cells to provide mechanical strength.
 Anchoring junctions are cell junctions that are attached
to one another and with the components of the
extracellular matrix. They are commonly found in tissues
that are prone to constant mechanical stress, e.g. skin
and heart.

3. Gap junctions-
 allow movement of molecules between cells
 At these junctions, the plasma membranes are not in actual
contact, but lie very close to each other, the gap being
reduced from the normal 20 nm to 3nm.

Glandular Epithelium
 The function of glandular epithelium is secretion, which is
accomplished by glandular cells that often lie in clusters deep
to the covering and lining epithelium.
 A gland may consist of one cell or a group of highly
specialized epithelial cells that secrete substances into ducts
(tubes), onto a surface, or into the blood.
 All glands of the body are classified as either endocrine or
exocrine.
 The secretions of endocrine glands enter the interstitial fluid
and then diffuse into the bloodstream without flowing
through a duct. These secretions, called hormones, regulate
many metabolic and physiological activities to maintain
homeostasis.
 The pituitary, thyroid, and adrenal glands are examples of
endocrine glands.
Exocrine glands
 Secrete their products into ducts that empty at the surface of
covering and lining epithelium such as the skin surface or the
lumen (interior space) of a hollow organ.
 The secretions of exocrine glands include mucus,
perspiration, oil, earwax, milk, saliva, and digestive enzymes.
 Examples of exocrine glands are sweat glands and salivary
glands.
 Some glands of the body, such as the pancreas, ovaries, and
testes, contain both endocrine and exocrine tissue.
 MEMBRANES
 Are flat sheets of pliable tissue that cover or line a part of the
body.
 The combination of an epithelial layer and an underlying
connective tissue layer constitutes an epithelial membrane.
 The principal epithelial membranes of the body are mucous
membranes, serous membranes, and the cutaneous
membrane, or skin.
 Another kind of membrane, a synovial membrane, lines
joints and contains connective tissue but no epithelium.

Mucous Membranes
 A mucous membrane or mucosa lines a body cavity that
opens directly to the exterior. Mucous membranes line the
entire digestive, respiratory, and reproductive systems and
much of the urinary system.
 The epithelial layer of a mucous membrane secretes mucus,
which prevents the cavities from drying out.
 It also traps particles in the respiratory passageways,
lubricates and absorbs food as it moves through the
gastrointestinal tract, and secretes digestive enzymes.
 The connective tissue layer (areolar connective tissue) helps
bind the epithelium to the underlying structures. It also
provides the epithelium with oxygen and nutrients and
removes wastes via its blood vessels.

Serous Membranes
 A serous membrane lines a body cavity that does not open
directly to the exterior, and it also covers the organs that lie
within the cavity.
 Recall that serous membranes consist of two parts: a parietal
layer and a visceral layer.
 The parietal layer- is the part attached to the cavity wall, and
the visceral layer is the part that covers and attaches to the
organs inside these cavities.
 Each layer consists of areolar connective tissue covered by
mesothelium.
 Mesothelium is a simple squamous epithelium. It secretes
serous fluid, a watery lubricating fluid that allows organs to
glide easily over one another or to slide against the walls of
cavities.

Synovial Membranes
 Synovial membranes line the cavities of some joints. They are
composed of areolar connective tissue and adipose tissue
with collagen fibers; they do not have an epithelial layer.
 Synovial membranes contain cells which secrete synovial
fluid. This fluid lubricates the ends of bones as they move at
joints, nourishes the cartilage covering the bones, and
removes microbes and debris from the joint cavity.
 General Features of Connective Tissue
 Connective tissue consists of two basic elements: cells
and extracellular matrix.
 A connective tissue’s extracellular matrix is the
material between its widely spaced cells.
 The extracellular matrix consists of protein fibers and
ground substance, the material between the cells and
the fibers.
 The extracellular matrix is usually secreted by the
connective tissue cells and determines the tissue’s
qualities. For instance, in cartilage, the extracellular
matrix is firm but pliable. The extracellular matrix of
bone, by contrast, is hard and not pliable.
 In contrast to epithelial tissues, connective tissues do
not usually occur on body surfaces.
 Also, unlike epithelial tissues, connective tissues
usually are highly vascular; that is, they have a rich
blood supply.
 Exceptions include cartilage, which is avascular, and
tendons, with a scanty blood supply. Except for
cartilage, connective tissues, like epithelial tissues, are
supplied with nerves.
CONNECTIVE TISSUE CELLS
 The types of connective tissue cells vary according to
the type of tissue and include the following:
 Fibroblasts- are large, flat cells with branching
processes. They are present in several connective
tissues, and usually are the most numerous.
 Macrophages- are phagocytes that develop from
monocytes, a type of white blood cell.
 Plasma cells- are an important part of the body’s
immune response.
  Mast cells- are involved in the inflammatory
response and also kill bacteria.
 Adipocytes-are fat cells.
 White blood cells are not normally found in significant
numbers in connective tissues. However, in response
to certain conditions, white blood cells can leave blood
and enter connective tissues.
 For example, neutrophils gather at sites of infection
and eosinophils migrate to sites of parasitic invasion
and allergic responses.

Connective Tissue Extracellular Matrix

 Each type of connective tissue has unique properties,
based on the specific extracellular materials between
the cells.
 The extracellular matrix consists of a fluid, gel, or solid
ground substance plus protein fibers.
Ground Substance
 Ground substance is the component of a connective
tissue between the cells and fibers.
 It supports cells, binds them together, and provides a
medium through which substances are exchanged
between the blood and cells.
 The ground substance plays an active role in how
tissues develop, migrate, proliferate, and change
shape, and in how they carry out their metabolic
functions.
 Ground substance contains water and an assortment
of large organic molecules, many of which are complex
combinations of polysaccharides and proteins.
 Another ground substance is the polysaccharide
chondroitin sulfate, which provides support and
adhesiveness in connective tissues in bone, cartilage,
skin, and blood vessels. Glucosamine is a protein–
polysaccharide molecule.

Fibers
 Fibers in the extracellular matrix strengthen and
support connective tissues.
 Three types of fibers are embedded in the extracellular
matrix between the cells: collagen fibers, elastic fibers,
and reticular fibers.
 Collagen fibers are very strong and resist pulling forces,
but they are not stiff (flexible), which promotes tissue
flexibility.
 These fibers often occur in bundles lying parallel to one
another. The bundle arrangement affords great
strength.
 Chemically, collagen fibers consist of the protein
collagen. This is the most abundant protein in your
body, representing about 25 percent of total protein.
 Collagen fibers are found in most types of connective
tissues, especially bone, cartilage, tendons, and
ligaments.
Elastic fibers
 Are smaller in diameter than collagen fibers, branch
and join together to form a network within a tissue.
 An elastic fiber consists of molecules of a protein called
elastin surrounded by a glycoprotein named fibrillin,
which is essential to the stability of an elastic fiber.
 Elastic fibers are strong but can be stretched up to
one-and-a-half times their relaxed length without
breaking.
 Equally important, elastic fibers have the ability to
return to their original shape after being stretched, a
property called elasticity.
 Elastic fibers are plentiful in skin, blood vessel walls,
and lung tissue.
Reticular fibers

 Consists of collagen and a coating of glycoprotein,

provide support in the walls of blood
vessels and form branching networks
around fat cells, nerve fibers, and
skeletal and smooth muscle cells.
 Produced by fibroblasts, they are much thinner than
collagen fibers.
 Like collagen fibers, reticular fibers provide support
and strength and also form the stroma (supporting
framework) of many soft organs, such as the spleen
and lymph nodes.
 These fibers also help form the basement membrane.
Classification of Connective Tissues
 The three broad categories of connective tissue are
classified according to the characteristics of their
ground substance and the types of fibers found within
the matrix.

I. Loose connective tissue

A. Areolar connective tissue
 B. Adipose tissue
C. Reticular connective tissue
II. Dense connective tissue
D. Dense regular connective tissue
E. Dense irregular connective tissue
F. Elastic connective tissue
III. Cartilage
G. Hyaline cartilage
H. Fibrocartilage
I. Elastic cartilage
IV. Bone tissue
V. Liquid connective tissue (blood tissue and lymph)
LOOSE CONNECTIVE TISSUE
 The fibers in loose connective tissue are loosely
arranged among the many cells.
 Lots of ground substance and cells. Fewer fibers.
 Leaves abundant empty spaces in tissue sections.
 The types of loose connective tissue are areolar
connective tissue, adipose tissue, and reticular
connective tissue.
Areolar connective tissue
 Description: It is not very well organized. One of the
most widely distributed connective tissues; consists of
fibers (collagen, elastic, and reticular) arranged
randomly and several kinds of cells (fibroblasts,
macrophages, plasma cells, adipocytes, mast cells, and
a few white blood cells) embedded in a semifluid
ground substance.
Combined with adipose tissue, areolar connective
tissue forms the subcutaneous layer, the layer of tissue
that attaches the skin to underlying tissues and organs.

Location: In and around nearly every body structure

(thus called “packing material” of the body);
subcutaneous layer deep to skin; superficial region of
dermis of skin; connective tissue layer of mucous
membranes; and around blood vessels, nerves, and
body organs.

Function: Strength, elasticity, and support.

Adipose tissue
Description:
 Has cells called adipocytes specialized for storage of
triglycerides (fats).
 Because cell fills up with a single, large triglyceride
droplet, cytoplasm and nucleus are pushed to
periphery of the cell.

Location
 Wherever areolar connective tissue is located;
subcutaneous layer deep to skin, around heart and
kidneys, yellow bone marrow, and padding around
joints and behind eyeball in eye socket.

Function
 Reduces heat loss through skin; serves as an energy
reserve; supports and protects organs
 It forms a cushion for shock absorption.

Reticular connective tissue

Description
 Fine interlacing network of reticular fibers (thin form of
collagen fiber) and reticular cells.

Location
 Stroma (supporting framework) of liver, spleen, lymph
nodes; red bone marrow, which gives rise to blood
cells; part of the basement membrane; and around
blood vessels and muscles.

Function
 Forms stroma of organs; binds together smooth
muscle tissue cells; filters and removes worn-out blood
cells in spleen and microbes in lymph nodes.

Dense Connective Tissue

Dense connective tissue- has fewer cells than loose.
Instead, its ECM is densely packed with collagen fibers.
Based on the arrangement of the fibers, there are two
subtypes of dense connective tissue; dense regular and
dense irregular.
Dense regular connective tissue- has the collagen
fibers aligned parallel to each other. This arrangement
provides the tissue with high unidirectional resistance
 to stress. The best dense regular connective tissue
examples are the tendons and ligaments.
Dense irregular connective tissue- has collagen fibers
randomly interwoven, forming a three-dimensional
network resistant to distension in all directions. It is
usually located in the capsules and walls of the organs,
the dermis of the skin and glands.
 Dense connective tissue contains more numerous,
thicker, and denser fibers (more closely packed), but
fewer cells than loose connective tissue.
 Dense connective tissue has poor blood supply which is
why tendon sand ligaments don’t heal quickly.
A. Dense regular connective tissue
Description:
 Dense regular connective tissue fibers are parallel to
each other, enhancing tensile strength and resistance
to stretching in the direction of the fiber orientations.
 Ligaments and tendons are made of dense regular
connective tissue, but in ligaments not all fibers are
 parallel. Dense regular elastic tissue contains some
elastic fibers in addition to the collagen fibers.
 Extracellular matrix looks shiny white; consists mainly
of collagen fibers regularly arranged in bundles;
fibroblasts present in rows between bundles.
 Collagen fibers are not living cells, but protein
structures secreted by fibroblasts, so damaged
tendons and ligaments heal slowly.

Location: Forms tendons (attach muscle to bone), most

ligaments (attach bone to bone), and aponeuroses
(sheetlike tendons that attach muscle to muscle or
muscle to bone).

Function: Provides strong attachment between various

structures. Tissue structure resists pulling (tension) along
long axis of fiber.

B. Dense irregular connective tissue

 In dense irregular connective tissue, the direction of
fibers is random. This arrangement gives the tissue
greater strength in all directions and less strength in
one particular direction.
 Consists predominantly of collagen fibers randomly
arranged and a few fibroblasts.
Location: Often occurs in sheets, such as fasciae
(tissue beneath skin and around muscles and other
organs), deeper region of dermis of skin, periosteum of
bone, perichondrium of cartilage, joint capsules,
membrane capsules around various organs (kidneys,
liver, testes, lymph nodes), pericardium of the heart;
also in heart valves.
Function: Provides tensile (pulling) strength in many
direction

C. Elastic connective tissue

Description: Consists predominantly of elastic fibers;
fibroblasts are present in spaces between fibers;
unstained tissue is yellowish.

Location: Lung tissue, walls of elastic arteries,

trachea, bronchial tubes, true vocal cords, suspensory
ligaments of penis, some ligaments between
vertebrae.
 Function: Allows stretching of various organs; is
strong and can recoil to original shape after being
stretched. Elasticity is important to normal functioning
of lung tissue, which recoils as you exhale, and elastic
arteries, whose recoil between heartbeats helps
maintain blood flow

CARTILAGE
 Cartilage consists of a dense network of collagen fibers
or elastic fibers firmly embedded in chondroitin
sulfate, a rubbery component of the ground
substance.
 Cartilage can endure considerably more stress than
loose and dense connective tissues. While the strength
of cartilage is due to its collagen fibers, its resilience
(ability to assume its original shape after deformation)
is due to chondroitin sulfate.
 The cells of mature cartilage, called chondrocytes,
occur singly or in groups within spaces called lacunae
in the extracellular matrix.
 The surface of most cartilage is surrounded by a
membrane of dense irregular connective tissue called
the perichondrium.
 Unlike other connective tissues, cartilage has no blood
vessels or nerves, except in the perichondrium.
 Cartilage does not have a blood supply because it
secretes an antiangiogenesis factor, a substance that
prevents blood vessel growth.
 Since cartilage has no blood supply, it heals poorly
following an injury.
 The three types of cartilage are hyaline cartilage,
fibrocartilage, and elastic cartilage.

Hyaline cartilage
 Contains the most abundant type of cartilage in the
body, consists of short and dispersed collagen fibers
and contains large amounts of proteoglycans. Under
the microscope, tissue samples appear clear.
 The surface of hyaline cartilage is smooth. Both strong
and flexible, it is found in the rib cage and nose and
 covers bones where they meet to form moveable
joints.
 It makes up a template of the embryonic skeleton
before bone formation. A plate of hyaline cartilage at
the ends of bone allows continued growth until
adulthood.
 Function: Provides smooth surfaces for movement at
joints, as well as flexibility and support.

Fibrocartilage
 Is tough because it has thick bundles of collagen fibers
dispersed through its matrix, which allows it to resist
compression and absorb shock.
 It is characterized by rows of chondrocytes.
 It is a very strong cartilage and thick collagen fibers.
 It is found in the intervertebral discs, pubic symphasis,
disk of knee joint.

Elastic cartilage
 Contains elastic fibers as well as collagen and
proteoglycans. This tissue gives rigid support as well as
 elasticity. The external ear and lid on top of larynx
(epiglottis) contains elastic cartilage.

Bone tissue

Muscle tissue
Muscle tissue has four main properties:
 Excitability: an ability to respond to stimuli
 Contractibility: an ability to contract
 Extensibility: an ability to be stretched without tearing
Elasticity: an ability to return to its normal shape
 Regardless of its morphology or type, muscle tissue is
composed of specialized cells known as muscle cells or
myocytes, commonly referred to as muscle fibers (all
of these terms are interchangeable); this is due to their
extensive length and appearance.
 Myocytes are characterized by protein filaments
known as actin and myosin that slide past one
another, producing contractions that move body parts,
including internal organs.
 At its most basic level, muscle tissue is classified as
either striated or non-striated/smooth based on the
presence or absence of ‘striations’ (i.e.stripes/furrows)
seen at a microscopic level; these are formed due to a
 particular arrangement of actin and myosin filaments
within the myocyte.
 Skeletal muscle is the most common type of muscle
tissue found in the body and consists of highly
elongated, multinucleate, non-branching cells which
are arranged in a parallel manner.

Skeletal Muscle Fibers

 Because skeletal muscle cells are long and cylindrical,
they are commonly referred to as muscle fibers (or
myofibers).
 Skeletal muscle fibers can be quite large compared to
other cells.
 Having many nuclei allows for production of the large
amounts of proteins and enzymes needed for
maintaining normal function of these large protein
dense cells.
 In addition to nuclei, skeletal muscle fibers also contain
cellular organelles found in other cells, such as
mitochondria and endoplasmic reticulum. However,
 some of these structures are specialized in muscle
fibers.
 The plasma membrane of muscle fibers is called the
sarcolemma and the cytoplasm is referred to as
sarcoplasm.
 Within a muscle fiber, proteins are organized into
organelles called myofibrils that run the length of the
cell and contain sarcomeres connected in series.
 The sarcomere- is the smallest functional unit of a
skeletal muscle fiber and is a highly organized
arrangement of contractile, regulatory, and structural
proteins.
 It is the shortening of these individual sarcomeres that
lead to the contraction of individual skeletal muscle
fibers.
 A sarcomere- is defined as the region of a myofibril
contained between two cytoskeletal structures called
Z-discs, and the striated appearance of skeletal muscle
fibers is due to the arrangement of the thick and thin
myofilaments within each sarcomere.
 The dark striated A band is composed of the thick
filaments containing myosin, which span the center of
the sarcomere extending toward the Z-dics.
 The thick filaments are anchored at the middle of the
sarcomere (the M-line) by a protein called myomesin.
 The lighter I band regions contain thin actin filaments
anchored at the Z-discs by a protein called α-actinin.
 The thin filaments extend into the A band toward the
M-line and overlap with regions of the thick filament.
 The A band is dark because of the thicker myosin
filaments as well as overlap with the actin filaments.
 The H zone in the middle of the A band is a little lighter
in color because it only contain the portion of the thick
filaments that does not overlap with the thin filaments
(i.e. the thin filaments do not extend into the H zone).
 Because a sarcomere is defined by Z-discs, a single
sarcomere contains one dark A band with half of the
lighter I band on each end.
 During contraction the myofilaments themselves do
not change length, but actually slide across each other
so the distance between the Z-discs shortens resulting
in the shortening of the sarcomere.
 The length of the A band does not change (the thick
myosin filament remains a constant length), but the H
zone and I band regions shrink. These regions
represent areas where the filaments do not overlap,
and as filament overlap increases during contraction
these regions of no overlap decrease.

Bone tissue
 Bone consists of four types of cells: osteoblasts,
osteoclasts, osteocytes, and osteoprogenitor (or
osteogenic) cells.
 Each cell type has a unique function and is found in
different locations in bones.
1. The osteoblast
 Is responsible for forming new bone, is found in the
growing portions of bone, including the periosteum
and endosteum.
 Osteoblasts do not divide.
 Synthesize and secrete the collagen matrix and
calcium salts. As the secreted matrix surrounding the
osteoblast calcifies, the osteoblast becomes trapped
within it. As a result, it changes in structure, becoming
an osteocyte.
2. Osteocyte
 are differentiated osteoblasts surrounded by
mineralized bone matrix
 Is the primary cell of mature bone and the most
common type of bone cell.
 Each osteocyte is located in a space (lacuna)
surrounded by bone tissue.
 Osteocytes maintain the mineral concentration of
the matrix via the secretion of enzymes.
 As is the case with osteoblasts, osteocytes lack
mitotic activity.
 They are able to communicate with each other and
receive nutrients via long cytoplasmic processes
that extend through canaliculi , channels within the
bone matrix.
3. The osteogenic cell
 These osteogenic cells are undifferentiated with
high mitotic activity; they are the only bone cells
that divide.
 Immature osteogenic cells are found in the deep
layers of the periosteum and the marrow. When
they differentiate, they develop into osteoblasts.

4.the osteoclast
 Is found on bone surfaces, is multinucleated, and
originates from monocytes and macrophages (two
 types of white blood cells) rather than from
osteogenic cells.
 Osteoclasts continually break down old bone while
osteoblasts continually form new bone.
 derived from the fusion of mononuclear hemopeiteic
cells (mononuclear phagocyte system)
 involved in resorption of bone matrix
 found in depressions on the bone surface of bone
tissue - Howship´s launae
 5-50 nuclei, very large cells - 40µm , motile cells
 abundant mitochondria - acidophilic cytoplasm
 great number of lysosomes
 Osteons/Haversian systems- are cylindrical
structures that contain a mineral matrix and a living
osteocytes connected by canaliculi.
 Each ring of the osteon is made up of collagen
and calcified matrix and is calles a lamella.

Blood
 Blood is made mostly of plasma, which is a yellowish
liquid that is 90% water.
 In addition to the water, plasma contains salts, sugar
(glucose), and other substances. And, most
important, plasma contains proteins that carry
important nutrients to the body’s cells and
strengthen the body’s immune system so it can fight
off infection.
 Blood is made mostly of plasma, but 3 main types of
blood cells circulate with the plasma:
 Platelets help the blood to clot. Platelets are also
called thrombocytes.
 Red blood cells carry oxygen. Of the 3 types, red
blood cells are the most plentiful. In fact, a healthy
adult has about 35 trillion of them. The body creates
these cells at a rate of about 2.4 million a second,
and they each have a life span of about 120 days.
 Red blood cells are also called erythrocytes.
 White blood cells ward off infection. These cells,
which come in many shapes and sizes, are vital to
the immune system.
 When the body is fighting off infection, it makes
them in ever-increasing numbers. Still, compared to
the number of red blood cells in the body, the
number of white blood cells is low.
 Most healthy adults have about 700 times as many
red blood cells as white ones. White blood cells are
also called leukocytes.
 Blood also contains hormones, fats, carbohydrates,
proteins, and gases.
What does blood do?
 Blood carries oxygen from the lungs and nutrients
from the digestive tract to the body’s cells.
 It also carries away carbon dioxide and all of the
waste products that the body does not need.
 Blood also helps keep your body at the right
temperature
 Carries hormones to the body’s cells
 Sends antibodies to fight infection