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Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain.

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Ultrastructural Distinction between Reticular and
Collagenous Fibers with an Ammoniacal
Silver Stain
Department of An at omy, Medical College of Vzrginia
R i ch mond, Virginza 23298
Reticular and collagenous fibers stain differently when subjected
to ammoniacal silver reduction. A variety of tissues were subjected to such a
"reticulin" technique and the association of reaction product with intercellular
connective tissue elements was studied with the electron microscope. The reaction with reticular fibers was primarily associated with the interfibrillar matrix,
and was globular i n form having a wide variety of particle sizes. Conversely,
in dermal collagen the unit fibrils were stained rather than the interfibrillar
matrix. The precipitate was punctate i n form and was associated with the cross
striations of unit collagen fibrils. Large microfibrils also reacted positively with
the stain, imparting a faint periodicity. Basement membranes were stained
uniquely. The underlying plasmalemma and the lamina densa were heavily
stained with silver while the lamina lucida was relatively unstained. The unit
fibrils of the lamina reticularis stained in the same manner as dermal unit collagen while the ground substance remained unstained. This represents a clear
distinction between the argentophilic characteristics of collagenous fibers, reticular fibers. and basement membranes
The network of extracellular connective
tissue (ct) fibers has attracted attention
since the earliest days of histology. The
pursuit of morphological and chemical distinctions among the classes of fibers lead
to the general acceptance of collagenous,
reticular and elastic fibers as the principal
fiber components of the ct space (Siegfried,
1892; Puchtler, '64; Foot, '27; Kramer and
Little, '53; Velican and Velican, '68; Ross
and Bornstein, '69; Stuart, '75). Microfibrils, slender filaments of 40-150 A diameter, have been demonstrated in transmission electron microscopy to be an integral
part of these ct fibers and have been postulated to be a formative precursor of unit
collagen fibrils (Low, '62, '68; Anderson,
'67; Morse and Low, '74), and elastic fibers,
(Ross and Bornstein, '69; Jones and Barson, '71). Recent studies have also demonstrated ontogenic relationships between
basement membranes and the fibrillar components of ct, especially collagenous and
reticular fibers (Low, '68; Fredrickson and
Low, '71; Matakas et al., '72; Carlson, '73;
Morse and Low, '74). Studies seeking to
distinguish between reticular and collagenous fibers have demonstrated that while
ANAT. REC-, 187: 191-206
their unit fibrils have many characteristics
in common (Kramer and Little, ' 5 3 ; Eastoe,
'67) the most striking distinction lies in the
composition of the matrix in which the respective fibrils are embedded (Glegg et al.,
'53; Irving and Tomlin, '54; Fernando et
al., '64). Reticular fibers were found by
Glegg et al. ('53), and by Windrum et al.
('55) to contain a large amount of carbohydrate compared to a very small amount
present in collagen (4% compared to
<0.5%). This carbohydrate is present as
a glycoprotein and acid hydrolysis of glycol
groups to free aldehyde groups followed by
oxidation with specific reagents is believed
to account for the positive periodic acid
Schiff (PAS) reaction, metachromasia after
sulfation metachromasia (Kramer and
Windrum, '55), and argentophilia of reticular fibers (Glegg et al.,'53) and basement
membranes (Marinozzi, '6 1 , de Martino
and Zamboni, '67).
Ammoniacal silver techniques have classically been used to demonstrate reticular
fibers. Collagenous bundles also stain with
ammoniacal silver although the reaction
Received Feb 23, '76 Accepted Sept 9, ' 7 6
product is much lighter and inore diffuse
than in reticular fibers. This difference
provides a basis for suggesting that reticular and collagenous fibers react in distinctive fashions. The present study was undertaken to explain at the ultrastructural
level of resolution differences in the silver
staining characteristics of these fibers
which are familiar to morphologists and
pathologists alike.
tion of aniline in glacial acetic acid (Lillie
and Glenner, '57) as a blocking reagent
for aldehydes. The samples were immersed
for 30 or 180 minutes prior to the acid
hydrolysis step of the silver stain.
Light microscopy
Reticular cells and their associated fibers
comprise the stroma of lymphatic tissue
and form a morphological basis of compartmentalization in mouse lymphatic tissue
(i.e., white and red pulp in the spleen).
This is demonstrated with silver reduction
techniques which selectively stain the
stroma. The adventitia of the central artery and the cortical (peripheral) region of
the white pulp nodule of the spleen contain a heavy concentration of reticular
fibers (fig. 1). Fibers of a variety of sizes
are present. The principal fibers stain darkly, and have numerous delicate branches
that form an interlacing net. This tissue,
as one observes in a hematoxylin-eosin
stained preparation, is populated primarily
with lymphocytes. At the LM level of resolution reticular fibers have a similar appearance in lymph nodes and in the parenchymatous organs such as the liver or pancreas. Darkly stained fibers also occur in
association with basement membranes,
such as those beneath the epithelium of a
hair follicle or the endothelium of a venule
(fig. 2).
Collagenous fibers stain weakly with silver reduction techniques. Consequently,
they appear brown in color when viewed
in the light microscope. Large bundles of
collagenous fibers are present in the dermis (fig. 2). Comparatively, the collagen
bundles stain lightly and lack lateral
branches analogous to those of reticular
fibers. Some variation in staining intensity
in the collagen can be seen depending on
the plane of section and orientation of individual fiber bundles.
Molar aniline in glacial acetic acid
blocked most of the fiber associated silver
reaction after 30 minutes immersion and
completely blocked the reaction after 180
minutes. Background staining associated
with cell cytoplasm and nuclei was not
The spleen, mesenteric lymph node, liver
and skin were removed from male BALB/c
mice and fixed in Tellyesniczky's fixative,
a formol-alcohol solution (Humason, '72),
or s-collidine buffered glutaraldehyde. In
preparation for chromatic staining or for
silver reduction, these tissue samples were
handled according to standard methods
for paraffin embedment. Serial tissue sections were stained with hematoxylin-eosin
or with Snooks reticulin stain (Luna, '68).
Small pieces ( < 1 mm3) of each tissue were
fixed for electron microscopy (EM) by immersion for four hours in 2.3% glutaraldehyde that was buffered to pH 7.2 with
0.06 M s-collidine. At this point, some tissue from each group was post-fixed in 1.3%
Os04 that was also buffered to p H 7.2 with
0.06 M s-collidine and prepared for conventional EM. The remainder was sliced
50-p thick pieces, as described previously
(Snodgrass and Peterson, '69), placed in
small vials and subjected to Snook's silver
reduction method for the demonstration of
reticular fibers (Rosen et al., '67; Luna,
'68). The tissue pieces were free floated
through Snook's technique, the various solutions being changed by Pasteur pipette.
The tissue pieces were then post-fixed
in s-collidine buffered Os04, dehydrated
through ascending concentrations of ethanol, propylene oxide and embedded in
Epon 812. Thin sections were prepared
with a Porter-Blum MT-2 microtome and
were mounted on 1 X 2 mm slot grids that
had carbon coated formvar membranes or
on naked 200-mesh copper grids. These
sections were studied with a Hitachi HU
12 electron microscope. Both unstained
sections and sections stained with uranyl
acetate and lead citrate were studied. Sections 1-p thick were stained with methyElectron microscopy
lene blue-azure I1 and studied with a Wild
M20 microscope.
The extracellular localization of splenic
Some samples were immersed in a solu- reticular fibers is shown in figure 3. The
unit fibrils are seen as small white structures, some cut in cross section and others
tangentially, which are embedded in a
dark matrix. Iqdividual unit fibrils range
from 200-250 A or more in diameter an$
possess a linear periodicity of 640-670 A.
Single reticular fibers vary from about 0.2
p to a maximum of 0.5 p in diameter and
possess highly variable numbers of unit
fibrils. Portions of the slender cytoplasmic
arms of splenic stromal reticular cells surround each individual fiber (fig. 3). This
cell-fiber association is typical in lymphatic
tissue, such as the spleen and mesenteric
lymph node, but not in parenchymatous
Ultrastructural distribution of the reaction product in tissues subjected to Snooks
reticular strain corresponds closely to the
distribution of reticular fibers (fig. 4). The
heaviest precipitation is at the periphery
of each reaction site and forms a ring
which surrounds several unit fibrils (figs.
4, 4 inset). Although this reaction obscures
some detail, its general pattern of distribution indicates that the reaction is associated primarily with components of the interfibrillar matrix rather than the unit
fibrils. Within the central portion of each
reaction site small granules show some
disposition for the matrix. but also appear
to have reacted with the unit fibrils. There
is, however, no distinct association of the
reaction product with subunits of these
fibrils, in which case the granules would
have been arranged in a cross banding
pattern. Outside of the dark rings of stain
there is no reaction product associated with
the cross bands of the unit fibrils (fig. 4)
as is the case with collagenous fibrils (fig.
5). The silver reaction with the interfibrillar matrix corresponds to the dark reticular fiber commonly seen with the light
microscope. Since only a portion of each
fiber is stained it is also evident that the
silver reaction does not necessarily reflect
the true size of any given reticular fiber.
Collagenous fibers differ from stromal
reticular fibers of lymphoid tissue in that
they may attain a diameter of two or more
micra and consist of a large number of
closely packed unit fibrils that range from
200-1,000 A in diameter. Also, the interfibrillar matrix of collagen fibers is structureless except for the presence of miaofibrils, which are generally about 80 A in
diameter. Collagenous fibers stain with
silver in a manner clearly different from
reticular fibers. Longitudinally sectioned
unit collagen fibrils from the dermis are
illustrated in figure 5 . The ag;ial macroperiodicity averages 6 4 0 4 7 0 A and is accentuated by the deposition of silver at the
major doublet of each repeating segment
of the unit fibril. A small amount of the
reaction product is also associated with
the minor cross bands. The quality of the
silver precipitate in the reticular fibers and
in collagenous fibers is compared in figures
4 and 5. The reaction associated with the
reticular fiber is globular and of variable
particle size making discrete, high resolution localization less apparent than in the
collagenous fibril. The precipitate associated with collagen has a small particle size
(20-50 A diameter) and is discretely associated with substructures of individual
unit fibrils. In cross sectioned unit collagen
fibrils particles of the silver precipitate
that are in the same 20-60 A size range
are present at both the surface and throughout the substance of each fibril (fig. 6). A
high degree of specificity of this reaction
is indicated by the lack of precipitate in
the connective tissue space adjacent to
these fibrils. Additionally, microfibrils, morphological subunits of the unit collagen
fibril, contain reactive components (fig. 7).
Silver particles are specifically associated
with microfibrils. Jel'
illustrated microfibril is about 150 A in diameter and very
small particles of silver either impart or
enhance a cross banding pattern. A similarly stained microfibril is present in figure 6.
Reticular fibers of the liver have a staining pattern that seemingly is intermediate
between that of the reticular fibers of lymphatic organs and the collagen of the derm i s (fig. 8). In the liver, silver particles
axe not present within the substance of
cross sectioned unit fibrils, which are electron lucent, but are associated more exclusively with the interfibrillar matrix.
This matrix is less dense than in lymphatic
tissue and the reaction is more generally
distributed. Although the reaction product
is larger than that found in collagen, it is
punctate in form. This is in contrast to the
globular reaction product seen in reticular
fibers of spleen and lymph nodes.
Basement membranes associated with
tissues such as epithelia and their derivatives, endothelium, and perineural and
Schwann cells of the peripheral nervous
system stain uniquely with Snooks silver
method (fig. 9). The plasmalemma of the
cell and the lamina densa of the basal lamina react vigrously, while the interposed
lamina lucida remains relatively unstained.
This staining pattern imparts a trilaminar
appearance. The unit fibrils of the lamina
reticularis stain as typical unit collagen
fibrils. Small punctate granules of silver
are deposited throughout the substance of
the transversely sectioned fibrils, while the
interfibrillar matrix remains unstained
(fig. 9).
After immersion of tissue samples in
molar aniline for 180 minutes the extrafibrillar localization of silver in reticular
fibers and the finely particulate precipitate
in unit collagen were not demonstrable
ultrastructurally. However, particulate deposits of silver were occasionally associated
with intracellular vesicular organelles and
The silver stain used in this study was
chosen because of its routine reproducibility in previous studies of lymphatic tissue
(Snodgrass and Hanna, '70; Snodgrass et
al., '72). The technique was recorded in
1944 (Snook, '44) and appears to have
evolved as an improvement of several earlier silver stains for reticulin (Kubie and
Davidson, '28; Pap, '29; Wilder, ' 35; Gordon and Sweets, '36). Similar techniques
have been used to demonstrate glycogen
(Mitchell and Wislocki, '44; Gomori, '46;
Arzac and Flores, '49). Arzac and Flores
('49) demonstrated that the spectrum of
reactivity of these stains for glycogen was
broadened to include reticulin when reduction with formalin was employed after
exposure to ammoniacal silver. They concluded that the silver was reduced by hepatic glycogen while being adsorbed to the
surface of reticulin. However, the subsequent demonstration of a respectable quantity of carbohydrate in reticulin (Glegg et
al., ' 5 3 ; Windrum et al.,'55) , and a small
amount of intrafibrillar carbohydrate in
collagen (Pease, '70; Pease and Bouteille,
'71) suggests that the silver is chemically
reduced within these fibers as well. Presumably this would have resulted from the
production of free aldehyde radicals by the
hydrolysis of glycol bonds in the carbohy-
drates present in the ct fibers and would
lead to the reduction of the ammoniacal
silver with precipitation at the reaction
site. This point of view is not in common
acceptance (Bloom and Fawcett, '75) but
is supported by the present study where
the reaction was observed within the matrix of reticular fibers and within the unit
fibrils of collagenous fibers.
Inhibition of the silver reaction in both
LM and EM preparations by immersion in
molar aniline in glacial acetic acid prior
to the acid hydrolysis and oxidation steps
of the silver stain is taken as strong evidence indicating that free aldehyde radicals were formed in the reticular and collagenous fibers.
In classical light microscopy the delicate,
highly branched reticular fibers stained intensely black with ammoniacal silver
stains, whereas coarse bundles of collagen
stained more lightly being brown to yellow
in hue. One quite logically assumed that
these differences resulted from the relative
deposition of elemental silver on each fiber
type. Indeed, ultrastructural studies of isolated unit fibrils that had been treated with
hyaluronic acid supported this view (Irving
and Tomlin, '54). However, it is apparent
from the present study that in intact tissue
the reaction in the two fiber types is not
morphologically analogous and that silver
particles are distributed three dimensionally throughout individual reticular fibers
and unit collagen fibrils. While there is a
general consensus that the unit fibrils of
reticular and collagenous fibers are very
similar (Gould, '68), morphological (Galindo and Freeman, '63; Fernando et al., '64;
Marinozzi, '61; Irving and Tomlin, '54) as
well as histochemical (Lillie, ' 5 2 ; P e a s e ,
'68) distinctions can be made. The reticular fibers of lymphatic tissue are unique
intercellular fibers that consist of electron
lucent unit fibrils that are generally of
smaller diameter than unit collagen fibrils
and are embedded in an electron opaque
matrix. When these fibers were stained with
silver the precipitate formed a ring within
the matrix and surrounded varying numbers of unit fibrils. While particles of stain
were associated with most unit fibrils within the reaction site, both the fibrils and
the matrix outside of the region of precipitation remained unstained. Reticular fibers
in the liver differ somewhat from those of
lymphatic tissue. They are not surrounded
by reticular cells and their matrix is less
electron opaque. Although the silver reaction is uniformly distributed throughout
individual fibers it is still preferentially
associated with the interfibrillar matrix
rather than the unit fibrils, again leading
to the conclusion that the majority of the
carbohydrate fraction of reticular fibers is
present in the interfibrilla matrix.
While the interfibrillar matrix of collagenous fibers remained unstained a positive
reaction was associated with both longitudinally and transversely sectioned unit
collagen fibrils. The reaction thus appears
to be distributed three dimensionally
throughout the unit fibril. Accepting the
tenet that this silver reaction demonstrates
the location of carbohydrate (Glegg et al.,
'53; Pearse, '68), one could conclude its
presence within the substance of unit collagen fibrils. This conclusion would be consistent with that of others (Pease, '66, '70;
Pease and Bouteille, '71) who have histochemically demonstrated a n intrafibrillar
carbohydrate matrix in unit collagen fibrils.
Large microfibrils in which faint periodicity could be seen also reacted positively
with the silver stain. This would point
towards the presence of a carbohydrate
moiety i n the formative elements as well
as in the mature unit collagen fibrils. Fredrickson and Low ('71) used a-amylase (a1,4 glucan 4-glucanohydrolase) digestion
to analyze various stages in the formation
of microfibrils. They concluded that an
amylase sensitive complex, which is interpreted here to mean a carbohydrate rich
complex, quite likely formed a template
for the condensation of soluble collagen
during the formation of microfibrils. The
morphological observations in this study
are at variance with the idea of the adsorption of silver to the surface of connective
tissue fibers. While reticular fibers, collagenous fibers, microfdaments and basement
membranes stain in distinctive fashions a
reasonable common explanation seems to
be the hydrolysis of 1,2 glycol bonds to aldehydes followed by the reduction of ammoniacal silver with precipitation at the
reaction site.
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This light micrograph demonstrates the silver impregnation of reticular fibers in a white pulp nodule of the spleen. The branched and interwoven nature of the fibers around the central arteriole (CA) and comprising the peripheral region of the nodule are prominent. X 125.
The heavy deposition of silver in basement membrane of cross sectioned
hair follicles and of the endothelium of a venule are contrasted with
the more lightly stained collagen fibers in the dermis. x 125.
This electron micrograph of splenic white pulp reticular fibers demonstrates the general axial orientation of the electron lucent unit fibrils
that are embedded in a dark matrix. These extracellular fibers are usually encircled by the slender arms of stromal reticular cells. X 15,000.
Michael J. Snodgrass
The densest precipitate i n this tangentially sectioned splenic reticular
fiber i s associated with the interfibrillar matrix. Small particles are
associated with both matrix and unit fibrils. x 65,000.
Inset. The reaction with this cross sectioned splenic reticular fiber
has a clearer predilection for the matrix, some unit fibrils within the
reaction site remain relatively unstained. X 105,000.
Michael J. Snodgrass
The principal site of reaction is at the major doublet which imparts
the 640 A macroperiodicity of unit collagen fibrils from the dermis.
Some silver particles also are associated with the minor cross bands
and with the matrix material at the surface of each fibril. X 160,000.
Michael J . Snodgrass
The punctate reaction product is distributed at the surface and throughout the substance of transversely sectioned unit collagen fibrils of the
dermis. X 75,000.
This mic;ograph illustrates the positive reaction of microfibrils. Large
particles of the silver precipitate are on the surface of this 150 A diameter microfibril, while small particles enhance its faint internal
cross banding pattern. x 210,000.
The matrix of reticular fibers in the liver is the primary reactive component. Transversely sectioned unit fibrils remain unstained. X 60,000.
A portion of a Schwann cell (S) and a perineural cell (P) with interposed endoneurium are present in this micrograph. The laminae densa
and the plasmalemmae, but not the laminae lucida, stained densely.
Unit fibrils of the lamina reticularis and adjacent ct space stain like
typical unit collagen fibrils (fig. 6). X 30,000.
Michael J . Snodgrass
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ultrastructure, fiber, silver, collagenous, distinction, reticular, ammoniacal, staib
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