Connective Tissue

Connective
and Muscle
Tissues
Mr:Mahmoud Ibrahim

Connective Tissue
– Connective tissue is 1 of the 4 basic tissues, and it
functions to provide structural and metabolic support for
the other tissues and organs in the body. It consists of 3
different components:
– fibers,
– cells,
– and amorphous ground substance.

– These components vary in amount within the different
connective tissue types, which are:
– 1. connective tissue proper
– 2. cartilage
– 3. bone
– 4. blood

– Most commonly, when we use special stains to
demonstrate connective tissue elements in the
histopathology laboratory, we are interested in the fibers
or cells of connective tissue proper.

1. Collagen fiber
– Provide strength; the more collagen present, the stronger
the tissue is. A dense regular arrangement of collagen
fibers is found in tendons, organ capsules, and
the dermis. At least 7 different types of collagen have
been identified with type 1 being the most common in
humans.

– The differences between the various types are
unnecessary for our understanding of the affinity of
collagen for certain stains. Collagen is very eosinophilic
and readily visible with light microscopy, is birefringent
upon polarization,

– Collagen is demonstrated using the Masson and Gomori
trichrome techniques, and with the van Gieson stain.

2. Elastic fibers
– are present in most fibrous connective tissue, but are most
abundant in tissue requiring flexibility, because the elastic
fibers allow tissues to stretch. The size and arrangement
– vary among different tissues, from fenestrated sheets or
– lamellae in the aorta to scattered fibers in loose connective
– tissue.

– These fibers usually cannot be seen on hematoxylin eosin
(H&E)-stained sections but require special stains, such as
the Verhoeff iron hematoxylin, Weigert resorcin fuchsin,
orcein, or Gomori aldehyde fuchsin stains, for
demonstration .

3. Reticular fibers
– have been identified as a type of collagen. These fibers
are not apparent in ordinary H&E-stained sections but
may be demonstrated with an argyrophilic reaction,
because they have the ability to adsorb silver from
solution.

– The silver may then be reduced chemically to its visible
metallic form.
– Reticular fibers form delicate networks and are much
smaller than most collagen fibers .

4 . Cells
– The cells found in connective tissue proper, whether fixed
, or free and transient, are:
– a. Fibroblasts, the most common cell in connective
tissue, which produce the connective tissue fibers
(extracellular, nonliving elements).

– b. Mesenchymal cells, which may be indistinguishable
from fibroblasts. These are primitive, relatively
undifferentiated cells that may develop into various
differentiated cell types if the need arises for replacement.

– c. Adipose, or fat, cells, which synthesize and store lipid
and are common in most loose connective tissue. In some
areas of the body, this is the predominant cell type and the
tissue is known as adipose tissue. The nucleus in an
adipose cell becomes very flattened as the lipid
accumulation of the cell grows.

– d. Mast cells, which contain abundant secretory granules
that, with special stains, frequently obscure the nucleus.
Mast cell granules contain histamine and heparin, and
exhibit metachromasia when stained with toluidine blue,

– Mast cells are most prominent along small blood vessels
and closely resemble the basophilic leukocyte found in
blood. Both mast cells and basophils can degranulate to
increase vascular permeability.

– e. Macrophages are "big eaters," or scavenger cells that are
found not only in connective tissue proper but in various other
tissues such as liver, myeloid, and lymphatic tissues.
Monocytes (blood leukocytes) are the precursor of
macrophages, and are also known as histiocytes

– f. Plasma cells, which are derived from B lymphocytes,
produce immunoglobulins. Before immunoenzyme techniques
were used for the demonstration of immunoglobulins, the
methyl green-pyronin stain was frequently used to help
identify immunoblastic sarcomas, a type of B-cell lymphoma.

– g. Blood cells of all types may be found in tissue

Basement Membrane
– The basement membrane, frequently referred to as the basal
lamina, is found beneath epithelium, and separates the
epithelium from the underlying connective tissue .The
basement membrane consists of type IV collagen, laminin (a
glycoprotein), and a proteoglycan rich in heparin sulfate

– Basement membranes are illustrated by techniques that
demonstrate the carbohydrate component. This is because
of both the glycoprotein present in the membrane and the
fact that the collagen present in the basement membrane
contains much more sugar in some of its side chains than
is normally present in ordinary collagen.

– The primary function of the basement membrane is to
provide physical support for epithelium; it also provides
for cell attachment and for ultrafiltration. In the kidney,
the basement membrane of capillary endothelium acts as
a sieve, holding back molecules on the basis of size,
shape, and electrostatic charge.

Muscle
– Muscle is also 1 of the 4 basic types of tissue and, based
on the differences in structure and in function, is
classified as follows:
– 1. Skeletal muscle. This type of muscle also may be
classified as:
– a. striated , because of the characteristic dark (A) and
light (I) bands seen microscopically, and
– b. voluntary, because contraction can be brought about at

– 2. Cardiac muscle. This is also a striated but involuntary
type of muscle. It is similar to skeletal muscle, except that
the cells branch and anastomose, and each cell usually
has only 1 centrally located nucleus.

– 3. Smooth muscle. This is a nonstriated, involuntary type
of muscle that is commonly arranged in layers. The
muscle fibers are long and tapered and contain a single
centrally located nucleus.

Staining Techniques for
Connective Tissue Fibers
– MASSON TRICHROME STAIN:
– Purpose
– Trichrome stains are frequently used to differentiate
between Collagen and smooth muscle in tumors and to
identify increases in collagenous tissue in diseases such
as cirrhosis of the liver.

– Principle
– Trichrome procedures are so named because 3 dyes,
which may or may not include the nuclear stain, are used.
The mechanism of the stain is not totally understood, and
may be related in part to the size of different dye
molecules. Sections are first stained with
– an acid dye such as Biebrich scarlet;

– all acidophilic tissue elements, such as cytoplasm,
muscle, and collagen, will bind the acid dyes. The
sections are then treated with phosphotungstic
and/or phosphomolybdic acid..

– Because cytoplasm is much less permeable than
collagen, phosphotungstic and phosphomolybdic
acids cause Biebrich scarlet to diffuse out of the
collagen but not out of the cytoplasm of cells

– Phosphotungstic and phosphomolybdic acid have
numerous acidic groups that most likely act as a
link between the decolorized collagen and aniline
blue, the collagen dye.

– Probably the pH of the
phosphotungstic/phosphomolybdic acid solution
also increases selective collagen staining and aids
in the diffusion or removal of Biebrich scarlet.

– Fixative Bouin solution is preferred, but 10% neutral-
buffered formalin may be used.
– Quality Control
– Practically every tissue has an internal control, so no
other control sections are needed; however, if a control is
desired, uterus, small intestine, appendix, or fallopian
tube will provide good material.

– Reagents
– Weigert Iron Hematoxylin Solution
– Biebrich Scarlet-Acid Fuchsin Solution
– Phosphomolybdic/Phosphotungstic Acid Solution
– Aniline Blue Solution

– Results:
– Nuclei Black
Cytoplasm, keratin, muscle fibers Red
– Collagen and mucin Blue

Technical Notes
– If desired, collagen may be counterstained with light
green instead of aniline blue.
– Light green is a better counterstain when collagen is
predominant, however, when only small amounts are to
be demonstrated, the aniline blue is the better
counterstain.

– Decreased red staining usually indicates that the
staining solution has aged or been overused and
should be discarded.

– If blue staining of connective tissue appears faded, the
section has probably been over differentiated in the acetic
acid solution. Pathologically altered collagen, such as that
seen in burns, may lose its affinity for aniline blue and
bind the acid dye instead.

– Sections fixed in 10% neutral-buffered formalin
will stain poorly and unevenly
– if not mordanted in either Bouin or a mercuric
chloride solution; however, mercuric fixatives
should not be used because of the toxicity.

Although most texts state that Weigert iron
hematoxylin should be prepared fresh, We find that
it is good for several days.

– An iron hematoxylin solution is used for nuclear
staining in the trichrome procedures because iron
hematoxylin is more resistant than aluminum
hematoxylin to decolonization in the subsequently
used acidic dye solution.

GOMORI 1-STEP TRICHROME
STAIN
– Purpose
– Identification of an increase in collagenous
connective tissue fibers or differentiation between
collagen and smooth muscle fibers.

– Principle:
– In the I-step trichrome procedure, a plasma stain
(chromotrope 2R) and a connective tissue fiber
stain (fast green FCF, light green, or aniline blue)
are combined in a solution of phosphotungstic
acid to which glacial acetic acid has been added

– Phosphotungstic acid favors the red staining of
muscle and cytoplasm. The tungstate ion is
specifically taken up by collagen, and the
connective tissue fiber stain is subsequently bound
to this complex.

– Fixative
– Any well-fixed tissue may be used. Bouin
solution is used as a mordant to intensify the
color reactions.

Technical Notes
– Sweat [1968] states that coloration of fine
connective tissue fibers is affected by the dye
solution pH, with maximum binding occurring
around pH 1.3. The pH of Gomori trichrome is
about 2.5, which decreases affinity for anions by
approximately 50%,

– so these investigators suggest that by replacing the
acetic acid with hydrochloric acid, a pH of
approximately 1.3 can be obtained. The intensity
of coloration of the fine connective tissue fibers
can be varied by altering the pH.

– Churukian [1993] finds that zinc formalin allows
good trichrome staining without mordanting in
Bouin solution

– Reagents
– Gomori Trichrome Stain
– Chromotrope 2R
– Fast green FCF, light green , or aniline blue
– Phosphotungstic acid
– Glacial acetic acid
– Distilled water

– Result:
– Nuclei Black
– Cytoplasm, keratin, muscle fibers Red
– Collagen and mucin Green or blue

VANGIESON PICRIC ACID-
ACID FUCHSIN STAIN
– Purpose
– Although the van Gieson technique may be
considered a primary connective tissue stain, it is
rarely used as such; however, it serves as an
excellent counterstain for other methods such as
the Verhoeff elastic technique.

– referred to in many institutions as the Verhoeff-
van Gieson (VVG) stain. Other institutions refer
to it as the elastic-van Gieson (EVG) stain.

Principle
– In a strongly acidic solution, collagen is
selectively stained by acid fuchsin, an acid aniline
dye. Picric acid provides the acidic pH necessary
and also acts as a stain for muscle and cytoplasm.
The low pH is very important, as selective
staining of collagen will not occur at higher pH
levels.

– The addition of 0.25 mL of hydrochloric acid to
100 mL of van Gieson solution will sharpen the
differentiation between collagen and muscle.
Saturated picric acid solutions are important in the
preparation of the stain and again in the selective
staining of collagen.

– Fixative
– Any well-fixed tissue may be used.
– Technical Notes
– An iron hematoxylin solution is used for nuclear staining in
– the trichrome procedures, because iron hematoxylin is more
– resistant than aluminum hematoxylin to decolorization in
– subsequent acidic dye solutions.

– If a sharp color differentiation is not obtained between
collagen and muscle, check the preparation of the
saturated picric acid solution, as the acidic pH provided
by this solution is very important differentiation.

– . Also the addition of 0.25 mL of hydrochloric acid to
lOO mL van Gieson solution may sharpen the color

– Reagents
– Van Gieson solution
– Acid Fuchsin, 1 % Solution (1 ml)
– Picric acid, Saturated Solution (9 ml)

– Results
– •Nuclei Black
– • Collagen Brilliant red
– • Muscle and cytoplasm Yellow

VERHOEFF ELASTIC STAIN
– Purpose
– Elastic fiber techniques are used for the demonstration of
pathologic changes in elastic fibers . These include
atrophy of the elastic tissue, thinning or loss that may
result from arteriosclerotic changes and reduplication,
breaks, or splitting that may result from other vascular
– Diseases.

– The techniques also may be used to demonstrate
normal elastic tissue, as in the identification of
veins and arteries, and to determine whether or not
the blood vessels have been invaded by tumor.

– Principle
– The tissue is overstained with a soluble lake of
hematoxylin-ferric chloride-iodine. Both ferric
chloride and iodine serve as mordants, but they also
have an oxidizing function that assists in converting
hematoxylin to hematein.

– The mechanism of dye binding is probably by
formation of hydrogen bonds, but the exact
chemical groups reacting with the hematoxylin
have not been identified. Because this method
requires that the sections be overstained and then
differentiated, it is a regressive method.

– Differentiation is accomplished by using excess
mordant, or ferric chloride, to break the tissue
mordant-dye complex. The dye will be attracted to
the larger amount of mordant in the differentiating
solution and will be removed from the tissue.

– The elastic tissue has the strongest affinity for the
iron hematoxylin complex and will retain the dye
longer than the other tissue elements. his allows
other elements to be decolorized and the elastic
fibers to remain stained.

– Sodium thiosulfate is used to remove excess
iodine. Although van Gieson solution is the most
commonly used counterstain, other counterstains
may be used.

– Fixative
– Any well-fixed tissue may be used, but neutral-
buffered formalin or Zenker solution is preferred.
– Quality Control
– Most laboratories use a section of aorta.

– Reagents
– Verhoff hematoxylin:
– Lugol Iodine
– Ferric Chloride, 10% Solution
– Alcoholic Hematoxylin, 5% Solution
– Ferric Chloride, 2% Solution (Differentiator)
– Van Gieson or Eosin Counter stain

– Results :
– • Elastic fibers Blue-black to black
– • Nuclei Blue to black
– • Collagen Red
– • Other tissue elements Yellow

Technical Notes
– It is easy to over differentiate this stain over
differentiated sections may be restrained at any
step
-Do not prolong staining with van Gieson solution,
because picric acid also will differentiate the stain
further.

– The preparation of van Gieson solution is critical
for proper differentiation of muscle and collagen.
If the picric acid is not saturated, collagen will
not stain red, and cytoplasm, muscle, and collagen
may all stain the same color

– For optimum results, slides must be individually
differentiated, because the time of differentiation
is somewhat dependent on the amount of elastic
tissue present. Do not depend on the control for
timing the differentiation of all sections.

ALDEHYDE FUCHSIN ELASTIC STAIN
[GOMORI, 1950, SHEEHAN 1980)
Purpose
– Refer to the Verhoeff elastic stain,
– Principle
– Hydrochloric acid and paraldehyde are added to an
alcoholic solution of basic fuchsin to form aldehyde
fuchsin

– The affinity of elastic fibers for this solution is not
understood. A number of other tissue elements
will also stain with aldehyde fuchsin. These
elements include pancreatic cell granules and
sulfated mucosubstances. Staining is intensified
by prior oxidation.

– Fixative
– 10% neutral-buffered formalin is preferred;
chromate fixatives should be avoided (why
Home work). Formalin- and Bouin-fixed tissues
will show a colorless background, and mercury-
fixed tissue will show a pale lilac background.

– Quality Control
– Use a section of aorta embedded on edge or a
cross-section of a muscular artery. Skin also
provides a good control.

– Solutions:
– Alcoholic Basic Fuchsin, 0.5% Solution
– Aldehyde Fuchsin Solution

Technical Notes
– The paraldehyde used for preparation of the
aldehyde fuchsin reagent should be fresh. Do not
use reagent that was opened previously.
– Old solutions of aldehyde fuchsin may not stain
well, and the staining time may need to be
prolonged.

Home work
– Mention other staining techniques of elastic
fiber

SILVER TECHNIQUES FOR
RETICULAR FIBERS
– Many variations of silver techniques can be used
for the demonstration of reticular fibers; the
principles, however, are the same for most of the
techniques and closely resemble those of the PAS
technique. The major steps are:

– 1. Oxidation of the adjacent glycol groups of the
hexose sugars in reticulin to aldehydes. Reagents
vary with the technique used, but the most
frequently used oxidizers are phosphomolybdic
acid, potassium permanganate, and periodic acid.

– 2. Sensitization, which usually is a metallic impregnation
step.
– Impregnation is the deposition of metallic salts on or
around the tissue element to be demonstrated. The exact
chemical reaction of sensitizers is not known, but
according to Sheehan and Hrapchak [1980], the metallic
salt used in this step probably forms a metal-organic
compound with the reticulin.

– The sensitizing metal is then replaced by silver. Commonly
used sensitizers are uranyl nitrate, ferric ammonium sulfate,
and dilute solutions of silver nitrate.
– 3. Silver impregnation involves treating tissue with an
ammoniacal or diamine silver complex, [Ag(NH) zl+. states
– that 4 atoms of silver will be deposited at the site of each
– reactive sugar residue in the reticulin

– and that the aldehyde groups present will reduce
the diamine silver to metallic silver; however, this
is not enough silver to provide adequate visibility.
– Further deposition occurs when incompletely
washed sections are transferred to formaldehyde.

– 4. Reduction uses formaldehyde in all methods. Residual
silver diamine ions are reduced to metallic silver by the
formaldehyde. The reduction step in silver impregnation
techniques is sometimes termed developing.

– 5. Toning is the term used when bound metallic silver is
treated with gold chloride, and the color of the
impregnated component is changed from brown to black.
The metallic silver is replaced by metallic gold in the
following reaction:
– 3Ag+AuCl3=Au +AgCl3

– A more stable compound is formed, and section contrast
and clarity are improved. The yellow color is removed
from the background by this step; however, toning can
be overdone, and a violet to red background instead of
the desired gray one will result.

– 6. Unreduced silver is removed by treating the sections
with sodium thiosulfate (hypo). This step will prevent
any nonspecifically bound silver remaining in the
section from being reduced by a later exposure to light.
– 7. Counterstaining may or may not be used, depending
on the type of tissue stained and personal preferences .

– Only 2 representative, reliable methods for reticular
fibers will be presented.

GOMORI STAIN FOR
RETICULAR FIBERS
– Purpose
– The demonstration of reticular fibers in tissue sections
can be important in the differential diagnosis of certain
types of tumors .
– A change from the normal reticular fiber pattern, as is
seen in some liver diseases, is also an important
diagnostic finding.

– Principle
– The hexose sugars of reticulin are demonstrated by
oxidation to aldehydes. Potassium permanganate is the
oxidizing agent in this procedure, and the excess is
removed by potassium metabisulfite. Ferric ammonium
sulfate acts as the sensitizer and is subsequently replaced
by silver from the diamine silver solution. Following

– impregnation, formalin is used to reduce the
silver to its visible metallic form. Follow with
toning with gold chloride and removal of
unreacted silver with sodium thiosulfate. The
final step is to counterstain, if desired.

Reagent's Snook Gordon and
Sweet
Gomori Laidlaw Nasher and
Shanklin
Wilder
Oxidizer Potassium
permanganate
Potassium
permanganate
Potassium
permanganate
Potassium
permanganate
Potassium
permanganate
Sulfuric acid
Sliver nitrate
Phosphomolybdi
c acid
Sensitizer Uranyl nitrate Ferric
ammonium
sulfate
Ferric
ammonium
sulfate
None Silver nitrate Uranyl nitrate
Impregnated
solution
Ammoniacal
silver
Ammoniacal
silver
Ammoniacal
silver
Lithium silver Ammoniacal
silver
Ammoniacal
silver
Reducing
solution
Formaldehyde Formaldehyde Formaldehyde Formaldehyde Formaldehyde
+Absolute
alcohol
Formaldehyde
+uranyl nitrate

– Quality Control
– Liver is a very good control tissue.

Technical Notes
– It is important that a hint of turbidity remain in the
silver solution. An excess of ammonia decreases the
sensitivity and results in incomplete impregnation
– of reticular fibers.

– The glassware must be chemically cleaned with
commercial cleaning agents or bleach. The older method
of using a mixture of sulfuric acid and potassium
dichromate is not recommended because of the hazards
involved in the preparation and use of this solution.

GORDON AND SWEETS STAIN
FOR RETICULAR FIBERS
– Purpose
– The demonstration of reticular fibers in tissue sections
can be important in the differential diagnosis of certain
types of tumors (Home work). A change from the normal
reticular fiber pattern, as seen in liver diseases such as
cirrhosis, hepatocellular fibrosis, and/or necrotic liver
disease, is also an important diagnostic finding.

– Principle
– The tissue is first oxidized by potassium permanganate
to enhance subsequent staining of reticular fibers , and
excess permanganate is removed by oxalic acid. Ferric
amsmonium sulfate acts as the sensitizer and is
subsequently replaced by silver from the diamine silver
solution. After impregnation, formalin is used to reduce
the silver to its visible metallic form.

– Before toning with gold chloride, unreacted silver
is removed with sodium thiosulfate. The final step
is to counterstain, if desired.
– Fixative
– 10% neutral-buffered formalin is preferred.

Staining Techniques for Muscle
– MALLORY PTAH TECHNIQUE FOR CROSS-STRIATIONS AND
FIBRIN:
– Purpose
– The demonstration of muscle cross-striations and fibrin . Cross
– striations are a diagnostic feature of rhabdomyosarcomas or
– tumors arising from striated muscle. Nemaline rods, (congenital,
hereditary neuromuscular disorder)

– present in some skeletal muscle diseases, may also be
demonstrated by the method. The PTAH has also been
used for the demonstration of glial fibers and myelin.
This method is rarely used today because it has been
replaced by immunohistochemical techniques.

Staining Technique for Basement
Membranes
– Periodic Acid Schiff Reagent (PAS):
– Purpose
– This procedure best delineates basement membranes, and is most
– often used in the histopathology laboratory for the detection of
– abnormalities or diseases manifested in the glomerular basement
– membrane.

– Principle
– The carbohydrate component of basement membranes is
oxidized to aldehydes by periodic acid. The aldehydes
formed by oxidation bind to the Schiff reagent give
magenta color

– Reference
– Histotechnology A self-Instructional Text 3 ed

– Thank for your attention
– For any question and to send me home work
– E mail: mahmoud19905@gmail.com
– WhatsAppssss : 0911621198

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