CONNECTIVE TISSUE

AND STAINS
INTRODUCTION

 Develops from the mesenchyme

 Consistss of cellular portion and a surrounding network of
non-cellular substances
 Cells include: fibroblasts, mast cells, histiocytes, adipose
cells, osteoblasts, chondrocytes, blood cells
 Intercellular substance contains amorphous and formed
elements
 Formed or fibrous types
 Amorphous or gel types
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Formed or Fibrous Intercellular
Substances
 Collagenic fibers
 Reticular fibers
 Elastic fibers

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COLLAGENIC FIBERS

 Most frequently encountered

 Appear as individual fibers or large bundles of fibers
 Strongly birefrigent in polarized microscope

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Collagen Type I

 Forms thick collagenous fibers that form the bulk

of collagen in the body
 Major structural protein in the lung

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Collagen Type II

 Found in hyaline and elastic cartilage produced by

chondroblast activity
 Fibers are thin with proteogylcans
 Not readily visible by light microscope
 Those that are found in articular cartilage are thicker and
resembles ulstracturally type 1 collagen
 Hyaluronidase is used to unmask type 2 to render
immunohistochemical evaluation

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Collagen Type III

 Found only on tissues with type I collagen

 Ex. liver, spleen, lung, kidney
 Referred to as fetal collagen (fetal skin contains 60% of
collagen type III compared to adults which is only 20%)

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Staining reaction of Collagen

 Type I collagen strongly stains with acidic dyes

due to cationic group of proteins

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RETICULAR FIBERS

 Fine and delicate fibers connected to collagen fibers

 Provide bulk of supporting framework of more cellular
organs such as spleen, liver, lymph nodes, where they
arranged in 3D network
 Shows weak birefringence under light microsopy
 May be demonstrated in paraffin sections using argyrophil-
type silver impregnation techniques or frozen section by
PAS

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ELASTIC FIBERS

 Found throughout the body especially associated with the

respiratory, circulatory and integumentary sytems
 Appears as fine single fibers (upper dermis) or membrane-
like structures (large arteries)

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Basement Membrane

 Resilient matrix that separates connective tissue from

epithelial, endothelial, or mesothelial cells, muscle cells,
fat cells, nervous cells
 Support epithelial surfaces, glands and other structures
like renal tubules, and endothelial cell linings of blood
vessels
 Difficult to distinguish using H&E technique
 Techniques used for critical examinations: PAS reaction
and oxidation-aldehyde demonstration techniques
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CONNECTIVE TISSUE STAINS

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TRICHROME STAINS

 A general name for number of techniques for the

selective demonstration of muscle, collagen
fibers, fibrin, and erythrocytes
 Example is the Van Gieson stain, Masson
Trichrome stain

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Factors Affecting Trichome Staining

 Tissue permeability and dye molecular size

 General rule in trichrome staining: smaller dye molecule
penetrate and stain a tissue element but whenever larger dye
molecule can penetrate the same element, the smaller molecule
will be replaced by it

 Heat - increase rate of staining and penetration of

dye molecule)
 pH- 1.5-3.0

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Nuclear Stains for Trichrome

 Iron hematoxylins are more preferred than alum

hematoxylins to prevent decolorization in the
subsequent staining with acid containing dyes
 Improved and resistant stain can also be done
using Celestine blue followed by conventional
alum hematoxylin

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Effects of Fixation

 Satisfactory fixatives for Trichrome staining: Zenker’s,

formal mercury, Bouin’s fixative or picro-mercuric
alcohol
 Tissues fixed in formaldehyde will not produce optimum
result with Trichome stains
 Thus, should be treated with picric acid and/or
mercuric chloride solutionto enhance intensity and
brilliance

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VAN GIESON’S STAIN
 Simplest method of differential staining of collagen using
a mixture of picric acid and acid fuschin
 Fixation: Mercuric chloride fixative

SOLUTION
Sat. aq. Picric acid solution 50 ml
1% aq. Acid fuschin solution 9.0 ml
Distilled water 50 ml

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VAN GIESON’S STAIN procedure

o Deparaffinize sections and bring to water

o Stain nuclei by celestine blue-hematoxylin sequence
o Wash in tap water
o Differentiate in acid alcohol
o Wash well in tap water
o Stain in Van Gieson solution for 3 minutes
o Blot and dehydrate through alcohols
o Clear in xylene and mount
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VAN GIESON’S STAIN results

o Nuclei- black
o Collagen-red
o Muscle, cytoplasm, RBC and fibrin- yellow

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VAN GIESON’S STAIN notes

o Fixation is not critcal- NBF is satisfactory

o Washing in water after Van Gieson’s should be avoided
o Nuclear staining should be intense- picric acid in Van
Gieson’s will act as differentiator
o For celloidin-embedded tissues, Weigert’s is
recommended than celestine blue

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MASSON’S TRICHROME STAIN

 Fixation: Zenker, Helly’s, Bouin’s, Formal sublimate or

formal saline
 Sections: All types

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MASSON’S TRICHROME STAIN results
o Nuclei- blue/black
o Cytoplasm, muscle and erythrocytes- red
o Collagen- blue

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MASSON’S TRICHROME STAIN procedure
 Deparaffinize and bring to water
 Remove mercuric pigment by iodine, sodium thiosulfate sequence
 Wash in tap water
 Stain nuclei by celestine blue or Wiegert’s
 Differentiate with 1% acid alcohol (when using celestine blue)
 Wash well in tap water
 Stain in acid fuschin for 5 minutes
 Rinse in distilled water
 Treat with PMA solution for 5 minutes
 Drain
 Stain with methylene blue (or light green)
 Treat with 1% acetic acid, 2 minutes
 Dehydrate, clear and mount

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MALLORY’s ANILINE BLUE

 Not differerential because other than staining collagen

fibers and reticulum it also stains hyalin fibrils, fibroglia
fibrils, smooth and striated muscle fibers and amyloid

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MALLORY’s ANILINE BLUE results

 Collagen fibrils, cytoplasm, fibroglia fibrils, axon cylinders

and neuroglia- red
 Elastic fibers- pale pink or yellow
 RBC and myelin sheath- yellow

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HEIDENHAIN’S AZAN TECHNIQUE

 Also known as Azocarmine stain- a modification

of Mallory’s Aniline blue
 Not recommended as general connective tissue
stain
 Useful in the demonstration of “wire loop lesions’
in the diagnosis of lupus nephritis in renal biopsies

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HEIDENHAIN’S AZAN TECHNIQUE
results
 Amyloid connective tissue and mucuos colloid-
deep blue
 Nuclei- red

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STAINS FOR DEMONSTRATION OF
FIBRIN
 Gram- Weigert
 Phosphotungstic acid-hematoxylin
 Trichrome methods
 (Martius, Scarlet ,Blue technique or MSB technique)

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STAINS FOR DEMONSTRATION OF
FIBRIN
•Gram- Weigert
•Phosphotungstic acid-hematoxylin
•Trichrome methods
•(Martius, Scarlet ,Blue technique or MSB technique)

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MSB technique for fibrin

 Standard technique employs

 Martius yellow- can be substituted with lissamine
fast yellow
 brilliant crystal scarlet- can be substituted with
Ponceau de xylidine or azofuschin
 soluble blue- can be substituted with durazol blue,
pontamine sky blue, fast green FCF and naphtalene
black 10B
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STAINS FOR ELASTIC FIBERS
•Verhoeff’s stain
•Taenzer- Unna Orcein method
•Weigert’s elastic tissue stain
•Masson’s trichrome stain
•Krajian’s method
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VERHOEFF’S STAIN

 Classical staining method for elastic fibers and

works well with all routine fixatives
 Satisfactory results may be obtained using
solutions up to 48 hour old

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VERHOEFF’S STAIN results

 Elastic tissue fibers- black

 Other tissues- depend on the counterstain used

 Sharpness and intensity of staining can be

improved with pre-treatment with 1% potassium
permanganate for 5 minutes

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Orcein method (Taenzer-Unna
Orcein)
 Excellent stain for elastic fibers- stains even the most
delicate and finest fibers of skin
 Main advantage: simplicity of preparation
 Primary stain: orcein
 Differentiator: acid alcohol
 Counterstain: methylene blue or alum hematoxylin

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Orcein method (Taenzer-Unna
Orcein) results
 Elastic fibers- dark-brown
 Nuclei- blue

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WEIGERT’S STAIN

 Primary stain: Weigert’s stain (Basic fuschin, resorcin

and ferric chloride)
 Differentiator: acid alcohol
 Counterstain: Neutral red, H&E, or hematoxylin and Van
Gieson’s
 Fixative: formalin or alcohol (Zenker fixed tissue stains
slowly)

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WEIGERT’S STAIN results

 Elastic fibers- dark blue or blue black

 Other structures- depend on the counterstain
used

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KRAJIANTECHNIQUE
 Rapid method of elastic tissue staining, for fibrin and
amyloid employing Congo red
 Elastic fibers- bright red
 Fibrin and connective tissues- dark blue
 RBC- orange yellow

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OTHER STAINS FOR ELASTIC
FIBERS
 Weigert’s resorcin-fuschin method
 Elastic fibers- brown to purple

 Methyl violet/ethyl violet resorcin method

 Elastic fibers- blue-black

 Aldehyde fuschin method

 Elastic fibers- blue-purple
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STAINS FOR RETICULAR FIBERS
•Metal impregnation techniques
•Gordon and Sweet’s method
•Gomori’s method
•Russel Modification of Movat Pentachrome stain

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SILVER IMPREGNATION TECHNIQUES

 Employ silver salts which is NOT visible with routine H&E

 Uses ammoniacal solution of Silver Carbonate reduced by
reticulum to dark brown Silver Oxide, in turn reduced to
black metallic Silver by formalin
 Unreduced Silver is removed by Sodium Thiosulfate
solution
 For complete permanent preparation: silver is partially
converted to gold impregnation by treatment with gold
chloride
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sections for reticular fiber SILVER
IMPREGNATION TECHNIQUES
 Frozen, cryostat, paraffin, and celloidin sections may be
employed for reticular fibers demonstration
 Fixatives containing salts of mercury or osmium cause
little non-specific background precipitation of silver

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GORDON AND SWEET’S METHOD for
reticular fibers
 Uses 5 ml of 10% aq.silver nitrate solution with
concentrated ammonia and 3% sodium hydroxide
 Notes:
 short treatment with iron alum solution, of <5 minutes, give
less staining of nuclei
 Use of Coplin jar for the silver solution reduces the
possibility of precipitation
 Sections can be counterstained with eosin, nuclear fast red,
tartrazine, or Van Gieson
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GORDON AND SWEET’S METHOD results

 Reticular fibers- black

 Nuclei- black or unstained
 Other elements- according to counterstain

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GOMORI’S METHOD for reticular
fibers
 Uses 10% potassium hydoxide solution with 40 ml of 10%
silver nitrate solution
 Results:
 Reticular fibers- black
 Nuclei- gray
 Other tissues- according to counterstain

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RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN
 Use for tissue fixed in 10% NBF or acetic formalin sublimate with tissue of 5
microns thick
 Employs:
 Solutions containing 1% alcian blue solution, alkaline alcohol, iodine solution, 10%
absolute alcohol hematoxylin, 10% ferric chloride
 Hematoxylin solution
 Crocein scarlet-acid fuschin

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RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN results
 Nuclei and elastic fibers- black
 Collagen and reticular fibers- yellow
 Ground substance, mucin- blue
 Fibrinoid, fibrin- intense red
 Muscle- red

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RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN notes
 Differentiation of elastic fibers is usually complete in 2-3
minutes
 Complete removal of alkaline alchol using runing water is
important, otherwise, subsequent staining will be
inhibited
 The stain may be used to demonstrate C. neoformans
staining brilliant blue

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OTHER STAINS FOR RETICULAR
FIBERS
 Bielchowsky’s Method
 Periodic Acid Schiff- Leucofuschin technique
 Gold method
 Laidlaw’s Silver impregnation
 Reticulum- violet to blue-black
 Snook’s reticulum method
 Reticulum- black

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