Connective tissue supports and connects organs and consists mainly of collagen, elastic fibers, and reticular fibers. Special staining techniques are used to identify these fibers, such as Masson's Trichrome to stain collagen blue and cytoplasm red, Gomori's to stain reticular fibers black, and Verhoeff's Van Gieson to stain elastic fibers blue-black. Connective tissue contains fibroblasts that produce fibers, as well as fat cells, mast cells, macrophages, and plasma cells. Muscle tissue includes skeletal striated muscle that is voluntary, cardiac muscle that is striated but involuntary, and smooth muscle.

1. York College CUNY
HPMT 332
Professor Emsley
Spring 2014
Connective Tissue

2. Definition of Connective Tissue
• Connective tissue may be defined as an
animal tissue that supports, connects, and
surrounds organs and other body parts
and consists mainly of collagen, elastic
fibers, fatty tissue, cartilage or bone.

3. Collagen Fibers
• Collagen-are the main protein of
connective tissue in animals and the most
abundant protein in mammals making up
about 25% of the total protein content.
• Collagen fibers provide strength; the more
collagen present, the stronger the tissue.
• A dense regular arrangement of collagen
fibers is found in tendons, organ capsules,
and the dermis.

4. Collagen contd.
• Collagen is eosinophillic and readily visible
with light microscopy.
• When we demonstrate connective tissue
element in histology we focus on the fibers
or cells of connective tissue proper.

5. Special Stains used to demonstrate
Collagen
• Trichrome stains are commonly 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.
• Trichrome procedures are so named
because 3 dyes which may or may not
include the nuclear stain, are used.

6. Masson Trichrome Technique
• Sections are first stained with an acid
dye such as Biebrich’s Scarlet ; all
acidophilic tissue elements, such as
cytoplasm, muscle, and collagen, will bind
the acid dyes.
• Next the sections are treated with
phosphotungstic and /or phosphomolybdic
acid.

7. Masson Trichrome Technique
contd.
• Because cytoplasm is much less
permeable than collagen, phosphotungstic
and phosphomolybdic acids cause the
Biebrich scarlet to diffuse out of the
collagen but not out of the cytoplasm.
• Controls are used during all special
staining technique.
• The control tests, that the stain is working.

8. Masson Trichrome Technique
contd.
• Results:
– Collagen, cartilage - blue
– Cytoplasm, muscle fibers- red
– Blood vessels – yellow, orange

9. Reticular Fibers
• Reticular fibers -are identified as one
type of collagen.
• Routine H&E stain doesn’t demonstrate
the fibers , but may be demonstrated with
an argyrophilic reaction, as they have the
ability to adsorb silver from solution.
• The silver may then be reduced to its
visible metallic form.
• Reticular fibers form delicate networks and
are much smaller than collagen fibers.

10. Reticular Fibers contd.
• Clean glassware has to be used because
silver is very peculiar.
• The Gomori’s technique is used for
demonstrating reticular fibers and is
important in diagnosing certain types of
tumors.
• A change in the normal reticular fiber
pattern gives an indication in the case of
some liver diseases.

11. Gomori’s Technique
• The tissue is first oxidized using
potassium permanganate to enhance
subsequent staining of the reticular fibers,
and the excess potassium permanganate
is removed with potassium metabisulfite.
• Ferric ammonium sulfate acts as the
sensitizer and is subsequently replaced
by silver from the diamino silver
solution.

12. Gomori’s Technique contd.
• Following impregnation, formalin is used
to reduce the silver to its visible
metallic form.
• Toning with gold chloride and removal of
unreacted silver with sodium thiosulfate
follow.
• Counterstain with nuclear fast red.
• The glassware has to be chemically
cleaned because silver is very peculiar

13. Gomori’s Technique contd.
• Results:
– Reticular fibers- black

14. Elastic Fibers
• Elastic fibers - are present in most
fibrous connective tissues, but are most
plentiful in tissue requiring flexibility as
the elastic fibers allow tissues to stretch as
in the aorta, largest artery.
• The fibers are not usually seen in routine
H&E and require special stain such as
Verhoeff’s van Gieson’s elastic technique.
• Elastic fiber techniques are used for the
demonstration of pathologic changes
in elastic fibers.

15. Gomoris One Step Trichrome
• Purpose: To identify an increase in collagenous
connective tissue fibers or differentiation
between collagen and smooth muscle fibers.
• Principle: plasma stain ( chromotrope 2R) and
connective fiber stain ( fast 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.

16. Staining Technique for One step
Trichrome
• 1 Deparaffinise and hydrate slides
• 2 Mordant in Bouins for 30 mins.
• 3 Wash well in water for 2 mins
• 4 Stain in hematoxylin for 2 mins
• 5 Rinse in water
• 6 Dip in ammonia for 10 secs
• 7 Stain in Gomoris 1-step trichrome for 15 mins
• 8 Rinse
• 9 Dehydrate 95 alc. Absolute, xylene then coverslip
• Result: nuclei – black, cytoplasm, muscle fibers- red,
collagen and mucin – green for fast green or blue for
aniline blue

17. Verhoeff’s van Gieson technique
• The tissue is overstained with a soluble
lake of hematoxylin-ferric chloride-iodine.
• Both ferric chloride and iodine served as
mordants, but they also have an oxidizing
function that assists in converting
hematoxylin to hematein.
• The sections are overstained and then
differentiated, so it is regressive.

18. Elastic Fibers contd.
• These include atrophy of the elastic
tissue, thinning or loss that may result
from arteriosclerotic changes and
reduplication, breaks or splitting that may
be used to demonstrate normal elastic
tissue, as in the identification of veins
and arteries, and to determine whether
or not blood vessels have been invaded
by tumor.

19. Verhoeff’s van Gieson technique
contd.
• 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 other tissue
elements.

20. Verhoeff’s van Gieson technique
contd.
• This allows other elements to be
decolorized and the elastic fibers to
remain stained.
• Sodium thiosulfate is used to remove
excess iodine.
• Van Gieson’s solution is the most
commonly used counterstain, but others
may be used.

21. Verhoeff’s van Gieson technique
contd.
• Results:
– Elastic fibers-blue-black, black
– Collagen- Red
– Nuclei-blue or black
– Other elements- yellow

22. Connective Tissue Proper
• Cells found in connective tissue proper- fibroblasts-most
common cell in connective tissue which produce
the connective fibers( extracellular, non living elements)
• Mesenchymal cells-
• Adipose or fat cells- synthesize and store lipid.
• Mast cells – contain secretory granules, histamine and
heparin
• Macrophages- “ big eaters” or scavenger cells, found
also in tissues eg. Liver, myeloid, and lymphatic tissues.
Play important role in immune mechanism.
• Plasma cells- derived from B lymphocytes, produce
immunoglobulins.
• Blood cells- of all types

23. Muscle
• 1. Skeletal muscle- may also be classified as
(a). striated- has the characteristic dark (A) and
light (I) bands seen microscopically, and (b)
voluntary- contraction can be brought about at
will.
• 2. Cardiac muscle- striated but involuntary type
of muscle, has specialized intercellular junctions
called intercalated discs.
• Refer to figs. (i8.1 Skeletal muscle and i8.2
Cardiac muscle