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Connective and mesenchymal tissues with their stains

Presentation · March 2019

DOI: 10.13140/RG.2.2.19815.91048

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Karam H Al-Mallah
University of Mosul
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Connective and mesenchymal
tissues with their stains

‫ كرم هاشم المالح‬.‫إعداد د‬

• Connective tissue is one of the four tissue types
• found throughout the body. The term ‘connect’
• comes from the Latin word ‘connectere’ meaning ‘to
• bind’. Its main function is to connect together and
• provide support to other tissues of the body. During
• embryonic development, the ectoderm and
endoderm are divided by a germ layer called the
mesoderm, generically known as mesenchyme. This
comes from the Greek words ‘mesos’ meaning middle
and ‘enchyma’ meaning infusion. It is from the
mesenchyme that the connective tissues develop.
• In almost any type of connective tissue there are
• three elements: the cells, the fibers, and the
amorphous ground substance.
• The connective tissues are
divided into the following
• groups:
• • Connective tissue proper –
includes loose or areolar,
dense, regular and adipose
irregular, reticular
• • Cartilage – hyaline elastic
and fibrocartilage
• • Bone – spongy or cancellous
and dense or
• cortical
• • Blood
• • Blood-forming –
hematopoietic.
• Connective tissue usually consists of a cellular
portion in a surrounding frame work of a non-cellular •
• The cell types of connective tissue can include entities
such as fibroblasts, mast cells, histiocytes, adipose cells,
reticular cells, osteoblasts and osteocytes,
chondroblasts and chondrocytes, blood cells, and
blood-forming cells. Some connective tissue has little
substance and consists primarily of cells whose
functions are not those of production of intercellular
substance, such as adipose tissue.
Intercellular substances may be readily classified into
two main groups by their microscopic appearance:
• • formed or fibrous types
• amorphous or gel types •
• A frequent fault among histologists is to speak of
• collagen, reticulin, and elastin, when in reality they
• mean collagenic fibers, reticular fibers, and elastic
• fibers. The former terms relate to the protein compound
• that is predominant in the particular fiber and should not
be used to describe the connective tissue fiber itself .

• Collagenic fibers
• These are the most frequently encountered of all the
• fibrous types of intercellular substance, and are
• found in large quantities in most sites in the body.
• They may occur as individual fibers, as in loose
• areolar tissues, arranged in an open weave system,
• or as large bundles of fibers clumped together to
• form structures of great tensile strength, such as
• tendon. Individual collagenic fibers are never seen
to branch •
 Types of collagen
Four major types of collagen and several minor
types have been characterized and described

• Type I
• This collagen forms the thick
collagenous fibers that
• have been demonstrated
histologically and form the
• bulk of the body’s collagen.
This type accounts for most of
the organic matrix of bases,
but is also a major structural
protein in the lung
Type II
This collagen is found in hyaline
and elastic cartilage and is
produced by chondroblast
activity.
Type III
This collagen is found only in
those tissues that also
contain Type I collagen (e.g. lung,
liver, spleen, kidney, etc.)
Type IV
This collagen has been
characterized in structures
identified morphologically as
basement membranes.

Types V and VI
Type V collagen is produced in
small quantities bya wide range
of cells, including connective
tissue cells, endothelial cells,
and some epithelial cells.
• Staining reactions of collagen
• Type I collagen stains strongly
with acid dyes, due
• to the affinity of the cationic
groups of the proteins
• for the anionic reactive groups of
the acid dyes.
• Collagen may be demonstrated
more selectively by
• compound solutions of acid dyes
(e.g. van Gieson)
• or by sequential combinations of
acid dyes
• (e.g. Masson’s trichrome,
Lendrum’s MSB, etc.) The
• different types of collagen may be
differentiated
immunohistochemically •
Reticular fibers
These are the fine delicate
fibers that are found
connected
to the coarser and stronger
collagenous fibers
(Type I fibers). They provide
the bulk of the supporting
framework of the more
cellular organs (e.g. spleen,
liver, lymph nodes, etc.),
where they are arranged in a
three-dimensional network to
provide a system of individual
cell support (gordon and
sweet staining)
• Elastic fibers
The elastic system fibers (i.e.
oxytalan, elaunin, and elastic fibers)
have, respectively, a fibrillar,
amorphous,or mixed structure. The
elastic fibers may be mostly
associated with the respiratory,
circulatory, and integumentary
systems. the elastic membranes are
interrupted by minute holes called
fenestrae (Latin ‘fenestra’ –
window) which permit diffusion of
materials through the otherwise
impermeable membrane They may
be seen in routine H&E- stained
sections, but, for exacting studies,
numerous more selective
techniques are available. These may
be relatively simple, e.g.
theTaenzer-Unna orcein method, or
more lengthy and complex, e.g.
Weigert resorcin-fuchsin methods.
With increasing age of the elastic
fibers
• Fibrin and fibrinoid
• Fibrin is an insoluble fibrillar protein formed by
• polymerization of the soluble fibrillar protein
fibrinogen, which is one of the plasma proteins.
• Fibrin is most commonly seen in tissues where
• there has been tissue damage, In paraffin sections
fibrin is strongly eosinophilic and stains blue-black
with Mallory’s phosphotungstic acid hematoxylin
(PTAH devised a trichrome method, the MSB, to
demonstrate fibrin and to attempt to distinguish
between fibrin of varying ages. ‘Fibrinoid’ is an
eosinophilic material which has identical staining
reactions to fibrin, but occurs in tissues in different
situations and disorders. It is frequently found within
vessel walls where the vessel has undergone acute
damage (‘necrotising vasculitis’), and sometimes as
plugs in capillaries
 Connective tissues
• The physical characteristics of the cells and the
intercellular substances vary considerably and they may
be divided into groups, according to the ratio of cells to
intercellular substance, and the types of cells and
• intercellular substance:
• areolar tissue
• adipose tissue
• myxoid connective tissue •
• dense connective tissue
• cartilage
• bone
• muscle – smooth, voluntary and cardiac
blood
Stains of connective tissues
 Gomori's hexamine silver (methenamine silver)
for basement membranes

Results:
Basement membranes - black. silver methamine fungal
Background - green. infection hyphe
 Gordon and Sweets' method for
reticulin
Results:
Reticulin - black.
Nuclei - red.
Collagen and cytoplasm - purple/pink.
 H.V.G. (Haematoxylin/van Gieson or
Weigert/van Gieson) for connective tissue
Results:
Nuclei - grey/black.
Collagen - red.
Muscle, red blood cells, cytoplasm -
yellow.
 M.P.T.A.H. (Mallory's phosphotungstic
Results: acid haematoxylin)
Nuclei, red cells, intracellular bridges of squamous epithelium,
cilia, mitochondria - blue.
Muscle striations, fibrin - dark blue.
Collagen, cartilage, bone matrix, reticulin, elastic fibres -
reddish brown.
Cytoplasm - pale brown to pink.
 M.S.B. (martius/scarlet/blue) for
connective tissue and fibrin
Results:
Nuclei - brown/black.
Collagen - blue.
Red blood cells - yellow.
Muscle and fibrin – red
 Masson's Trichrome Stain
Results:
Nuclei - Black
Cytoplasm, keratin, muscle fibres,
intercellular fibres - red.
Collagen, mucins - blue (or green).
 Miller's elastin stain (E.V.G.)
Results:
Elastin and mast cell granules -
black/purple.
Collagen - red.
Muscle and cytoplasm - yellow.
 Sudan black method for lipid
Oil red O for fats (frozen sections only) Lipid appears in black
Results:
Lipids - bright red.
Nuclei - blue.
 Safranine O/fast green for cartilage
Results:
Nuclei - black.
Cytoplasm - green.
Mucins, cartilage and mast cell granules -
orange/red.
 Orcein stain for elastic fibers
Orcein staining of liver tissue. The elastic fibres
in the portal tract are a deeper brown (left).
Similarly, the copper-associated protein in the
hepatocytes adjacent to the portal tract are a In skin
similar deep brown.
Weigert’s resorcin-fuchsin Verh eff’s method for
method for elastic fibers elastic fibers
Results Elastic tissue fibers black
Elastic tissue fibers brown to purple Other tissues according to counterstain
Heidenhain’s ‘Azan trichrome stain
Collagen =violet , cartilage=blue
cytoplasm = pink , nuclei= magenta
 Russell Movat Pentachrome Stain Kit
Stains all connective tissue elements, mucins, and
nuclei in just over one hour.

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