0022-1 554/85/$3,30

The Journal of Histochemistry and Cytochemistry Vol. 33, No. 8, pp. 737-743, 1985
Copyright 1985 by The Histochemical Society, Inc. Printed in U.S.A.

Original Articles

A Simple Micromethod for Collagen and Total

Protein Determination in Formalin-fixed
Paraffin-embedded Sections12

ALFREDO LOPEZ-DE LEON and MARCOS ROJKIND

Departamento tie Bioqulmica, Centre tie Investigaci#{243}n y tie Estudios Avanzados, I.P.N., Mexico, D.F. 07000

Received for publication January 26, 1984 and in revised form August 7, 1984 and February 5, 1985; accepted
February 15, 1985 (4A0018)

A simple, sensitive, and quantitative procedure is de- sections and measuring the collagen content by hydrox-
scribed for the measurement of collagen and protein con- yproline analysis and the amount of protein by the micro-
tent in tissue sections prepared from formalin-fixed par- Kjeldahl procedure. When several sections prepared from
affin-embedded samples. The method can detect as little five rat tissues were analyzed first by the dye binding method
as 5.7 ag of collagen per mg of protein. It is based on the and then by the chemical procedure, comparable results
selective binding of Sirius red F3BA and Fast green FCF were obtained. This method could be of use in measuring
to collagen and noncollagenous components, respectively, collagen in tissue specimens and could be helpful in as-
when the sections are stained with both dyes dissolved in sessing the degree of fibrosis in tissue samples and in eval-
aqueous saturated picric acid. Both dyes are eluted readily uating the effects of antifibrogenic drugs currently in use.
and simultaneously with NaOH-methanol and the absor- KEY WORDS: Sirius red F3BA; Fast green FCF; Collagen quart-
bances obtained at 540 and 605 nm can be used to deter- titation; Protein quantitation; Tissue sections; Histochemistry;
mine the amount of collagen and protein. The color equiv- Dye binding.
alence of each dye was determined after destaining the

der to obtain greater accuracy in assessing the degree of fi-

Introduction brogenesis from the noninvasive methods.
A liver biopsy is a simple and relatively safe procedure that We have recently shown that Sirius red F3BA can be used
can be used routinely to establish the diagnosis ofchronic liver for the quantitative estimation of collagen chains separated by
diseases and cirrhosis. However, the method cannot be used sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis
frequently enough to follow the progression of chronic liver (1). This dye has been used since 1964 for staining collagen
disease to cirrhosis, or to evaluate the beneficial effects on in histologic specimens (25), and has been recently applied in
fibrosis of drugs such as colchicine (14) and penicillamine (4), measuring soluble collagen that had been fixed to glass slides
which are currently in use for the treatment of liver cirrhosis. (9) or the amount of collagen remaining after incubation of
Several noninvasive methods have been developed as pos- the protein with collagenase (15,19).
sible markers of liver fibrosis. They are based on the analyses We now report a simple and reproducible method that can
in serum of enzymes involved in posttranslational modifica- be used routinely to estimate collagen concentration in for-
tions of collagen (23). Because of the lack of quantitative maim-fixed paraffin-embedded sections. The method is based
methods to measure collagen in liver biopsies, serum enzyme on the selective binding of Sirius red F3BA and Fast green
values are compared with arbitrary scores derived from qual- FCF to collagen and noncollagenous proteins, respectively.
itative estimations of the degree of inflammation and/or fibro- The dyes have maximal absorbances at different wavelengths
sis. Therefore, a simple and reproducible method is needed and can be eluted simultaneously and quantitatively with
to determine collagen concentration in biopsy material in or- NaOH-methanol from histologic sections.

Supported in part by grant 283-0043 from the Edna McConnell Materials and Methods
Clark Foundation.
2This work was presented at the 34th annual meeting of the Amer- All the chemicals used were of commercial origin and of the best
ican Association for the Study of Liver Diseases and appeared else- grade available. Sirius red F3BA was purchased from Mobay Chemical
where in abstract form (16): Company, Dye Stuff Division; Fast green FCF (color index 42053)

737
738 LOPEZ-DE LEON, ROJKIND

was obtained from Allied Chemical Corporation. All the tissue sec- ing was performed as described by Armend#{225}riz-Borunda and Rojkind
tions used were obtained as follows: male Wistar albino rats, weighing (1), except that Sirius red F3BA was prepared with Fast green FCF
approximately 200 g, were decapitated and sections of liver, heart, as described in this article. Samples were destained with metha-
lung, spleen, and kidney, 3 mm thick each, were cut with a razor blade nol : acetic acid : water (30 : 7 : 63 ; v : v : v) and densitometric tracings at
and immediately fixed in 10% formalin in 0. 1 M phosphate buffer, 540 and 605 nan were obtained with a Beckman 35 spectrophotometer.
pH 7.2, that contained 0.15 M NaCI. Samples were embedded in
paraffin and sections, approximately 15 j.tm thick, were obtained. Groups
of 10 to 20 sections were deparaffinized after incubation with xylol, Results
xylol : ethanol ( 1 : 1 ), ethanol, water : ethanol ( 1 : 1 ), and water. Samples
The procedure herein described was applied only to formalin-
were kept in distilled water at 4#{176}C
and used during the course of
fixed samples and therefore its use with other fixatives will
1 week.
have to be investigated. However, liver samples fixed with
Staining procedure. Individual slices, containing a sectional area Bouins solution gave equivalent results to those obtained with
of approximately 30-50 mm2, were placed in small test tubes (10 x
formalin-fixed tissues GA Grimaud and M Rojkind, unpub-
75 mm) and covered with 0.2 ml of a saturated solution of picric acid
lished results).
in distilled water that contained 0. 1% Fast green FCF and 0. 1% Sirius
To develop a quantitative method for collagen estimation
red. The tubes were covered with aluminum foil and incubated at
room temperature for 30 mm in a rotary shaker. Fluids were carefully
in tissue sections, it was important to have a second quanti-

withdrawn with a disposable pipette and the sections were rinsed tative parameter to which to refer the collagen values obtained.
several times with distilled water until the fluid was colorless. One Because the sections used had been already fixed in formalin
milliliter of 0. 1 N NaOH in absolute methanol (1 : 1, v : v) was then and embedded in paraffin, their weight was completely un-
added and each tube gently mixed until all the color was eluted from reliable. Therefore, we investigated the possibility of using the
the section (usually within a few seconds). The eluted color was read protein content of the sections to express the collagen values
immediately in a Beckman 35 spectrophotometer at 540 and 605 nm, as a function ofprotein concentration. Initially we used Coom-
i.e. , the wavelengths corresponding to the maximal absorbances of
assie blue G-250 because the dye has been used extensively
Sirius red F3BA and Fast green FCF, respectively. The sections were
for protein determination (3). However, the strong meta-
saved for collagen and protein estimations, vide infra. The eluted
chromatic behavior of the dye (27) and its nonspecific aggre-
colors were stable for at least 7 days when kept in the dark at 4#{176}C.
Because Fast green FCF was shown to contain a shoulder with
gation on tissue sections precluded its use.
absorbance at 540 nm, a standard curve with different concentrations Fast green FCF was shown to be a good dye for determining
of the dye was performed in order to determine the contribution of noncollagenous protein content in tissue sections. A solution
Fast green FCF to the overall absorbance of Sirius red F3BA at of Fast green FCF in picric acid stained the sections homo-
540 nm. geneously and did not interfere with the staining of collagen
Protein and hydroxyproline determinations. Duplicate or trip- fibrils by Sirius red F3BA. The amount of dye fixed to the
licate slices, saved after destaining, were placed inside glass vials that tissue was proportional to the thickness of the sections when
contained 0.5 ml of 6 N HCI; they were sealed under vacuum and they were derived from the same paraffin block (same sec-
hydrolyzed for 18 hr at 104#{176}C.Excess HCI was removed under vacuum tioned area). Depending on the tissue used, the amount of
and the dried sample was resuspended in 0.5 ml of2 N HCI, incubated color bound increased with time and leveled off at various
with Dowex-Norit to remove humin (2 1 ), neutralized to pH 5-6 with times before 30 mm of incubation. Prolonged incubation, up
NaOH, and adjusted with distilled water to a final volume of 2 ml.
to 2 hr, did not increase the amount of dye bound to the
Aliquots containing 0.2 and 0.5 ml were used for hydroxyproline
tissue. When sections were stained first and then used for
determination according to the procedure of Rojkind and Gonzalez
protein determination by micro-Kjeldahl analysis, the values
(22). One milliliter ofthe solution was used for nitrogen determination
obtained for the color eluted were always proportional to the
by micro-Kjeldahl analysis (17). For collagen measurements, it was
assumed that one a chain of approximately 96,000 daltons contains protein content of the section.
on the average 100 residues of 4-hydroxyproline. For protein esti- In a series of experiments, sections were first stained with
mation, it was assumed that proteins in the tissues investigated were Fast green FCF and then with Sirius red F3BA, and others
composed of 16% nitrogen. Because the amount of collagen in the were stained simultaneously with both dyes. Since the absor-
five tissues analyzed was relatively small (>6.5%), no corrections were bances of the eluted colors at 540 and 605 nm were the same,
made in the calculations of color equivalences for the noncollagenous independently of the order of incubation with the two dyes,
proteins (see below).
all further experiments were performed with the mixture of
Once the color equivalences were obtained, new tissue specimens
Fast green FCF and Sirius red F3BA in saturated picric acid.
were processed to compare the results using both chemical and dye-
However, it is important to emphasize that picric acid must
binding procedures. For this study, tissue samples weighing approx-
imately 250 mg (wet weight) were used for analysis by the chemical be present at all times, since interaction of Sirius red F3BA
method and multiple sections were analyzed by the dye-binding pro- with collagen is dependent on its presence. In rat liver and
cedure. A total of four animals were used for this study. spleen, a 1 5 mm preincubation with a 0. 1 % solution of Fast
green FCF in saturated picric acid was helpful in preventing
Acrylamide gel electrophoresis. In order to check whether all
the collagens stained with Sirius red F3BA and that there was no
staining with Fast green FCF, collagens from lung, spleen, and kidney Figure 1. Photomicrographs of6 tm sections stained with 0. 1% Sirius
biomatrices were solubilized with pepsin (1) and aliquots containing red, 0. 1% Fast green in a saturated solution of picric acid in water.
50 to 100 j.Lg of collagen determined by hydroxyproline analysis (22) Both interstitial (ABF) and basement membrane collagens (CDE) stain
were applied on 5% SDS-acrylamide gels. Electrophoresis and stain- with Sirius red F3BA. Original magnification x 100. Bar = 40 .tm.
 . - -. .- . , i,.- , -
l4 I,

..1i
1
., .
.-.

,.
: .

-:

, .*yf ., :, #{163}

c .

- 4.) - .
V.

4.
,.
, - .

-.-.
4 ., ,-#{149} 5#{176}

p .:.:-. -
p..

t .4,

Figure 1
740 LOPEZ-DE LEON, ROJKIND

any nonspecific staining with Sirius red F3BA; without this

treatment,
red also.
the nuclei ofhepatocytes and lymphoid cells stained
1.4 -
Figure 1 depicts the staining pattern obtained with different
tissues. Collagens that stain red have a fibrillar appearance. 1.2 -
The noncollagenous components that stain green have a more
Ui
diffuse
amounts
pattern.
of the
Because
specific
the
genetic
different
types
tissues
of
contain
collagen,
different
it was im-
C) 1.0 -
z
portant to determine if all collagens were absorbing the dye,
4
and also, if the color equivalences for all tissues were similar. 0.8 -

As illustrated in Figure 1A, B, and F, the interstitial collagens

of the portal tracts,
and the interstitial
the reticulum
fibers of the
fibers of the liver
heart muscle stain
and spleen,
a deep red;
0
(1) 0.6 -
thus it would seem that interstitial collagens I and III
(2,5,6,7,13,26) absorb the Sirius red F3BA dye. In addition,
4

0.4 -
as illustrated in Figure 1 C, D, and E, the basement membranes
of the glomeruli, tubuli, and bronchi (1 8) also stain with Sirius
red F3BA. The fine fibrils of kidney mesangium (Figure 1C), 0.2
possibly type V collagen (2), also stain red. These results sug-
gest that at the histochemical level, both interstitial as well as I I I I I I I
C-
basement membrane collagens absorb the red dye. However, 400 450 500 550 600 650 700
no quantitative data can be generated from these findings. WAVELENGTH (nm)
In order to further show that collagens do not absorb sig- Figure 3. Absorption spectrum of Fast green eluted from a tissue
nificant amounts of Fast green FCF dye and that they stain section that was stained with 0. 1% Fast green FCF dissolved in a
selectively with Sirius red F3BA, the electrophoretograms per- saturated solution of picric acid in water. The color was eluted as
described in Figure 2.
formed with collagens obtained from kidney, spleen, and lung
(tissues known to contain variable amounts of interstitial and
basement membrane collagens) were stained with the mixture
absorbing at 605 nm was detected in the region of the al
of the two dyes. The densitometric tracings performed after
chain of type I collagen (not shown).
destaining revealed that all the collagens contained material
The absorption spectrum of Sirius red F3BA is shown in
with an absorbance at 540 nm and only a trace of material
Figure 2. It has a peak of maximal absorbance at 540 nm. As
illustrated in Figure 3, Fast green FCF has its maximal absor-
Figure 2. Absorption spectrum of Sirius red eluted from a tissue bance at 605 nm, but contains a shoulder in the 540 nm region.
section that was stained with Sirius red F3BA dissolved in a saturated Since this shoulder could interfere with collagen measure-
solution of picric acid in water. The color was eluted with 1 ml of a
ments, it was important to determine its overall contribution
1 : 1 mixture (v : v) of 0. 1 N NaOH-absolute methanol.
at that particular wavelength. Solutions containing different
concentrations of Fast green FCF were scanned and the relative
0.7 -
absorbances at 540 and 605 nm were determined. It was ob-
served that the absorbance at 540 nm was proportional to that
obtained at 605 nm and was found to be 29. 1 0. 16%. Sirius
0.6 - red, however, has no absorbance at 605 nm and, therefore,
has no interference with noncollagenous protein determina-
tion. Figure 4 depicts the absorption spectrum ofthe combined
0.5 -

dyes that were eluted with NaOH-methanol from a stained

cirrhotic liver section. As can be seen in the figure, the peaks
of maximal absorbance have sufficient separation to allow the
measurement of collagen and noncollagenous proteins.
The color equivalences were determined from the rela-
tionships between the absorbances obtained with the eluted

< 0.2 - colors

performed
and the chemical
with the
measurements
destained sections.
ofcollagen
Tables
and protein
1 and 2 depict
the typical results obtained with sections prepared from a sin-
0.1 gle animal using the same paraffin block. As can be seen in
Table 1 , the color equivalence obtained for Fast green FCF
was 2.04 0. 18 and was very similar for all the tissues in-
400 450 500 550 600 650 700 vestigated. The color equivalence obtained for Sirius red F3BA
WAVELENGTH (nm) was approximately 20-fold greater than that of Fast green FCF,
MICROMETHOD FOR COLLAGEN DETERMINATION IN TISSUE SECTIONS 741

Table 2. Color equivalence ofcollagen as determinedfrom the

binding ofSirius red

.4 - Corrected absorbance Collagen content Color

Tissue at 540 nm (zg/section)b equivalence

Liver 0.346 0.032 8.59 39.7 1.30

Ui
Kidney 0.267 0.049 6.97 37.3 3.63
C) Lung 0.240 0.005 663 36.3 0.73
z Heart 0.111 0.011 3.13 35.6 2.05
4 Spleen 0.181 0.032 4.50 40.2 3.70

Average 37.8 2.05

0
U) Mean absorbance ( SD) of 5 to 10 sections obtained from a single animal.
The values were corrected by subtracting the 29. 1% contribution of Fast green
4 at 540 nm. These sections were used for collagen determination by hydroxy-
proline analysis.
5Collagen concentration was determined by hydroxyproline analysis of pools
of 3 to 4 sections obtained after elution of the color with NaOH-methanol.

:.::
Values are means.
The color equivalence is expressed as corrected OD at 540/mg of collagen.
Values are means SD.

400 450 500 550 600 650 700 F3BA dye, it was concluded that within experimental error,
WAVELENGTH (nm) all collagen types bind similar amounts of Sirius red. There-
fore, the method can be applied for measuring collagen content
Figure 4. Absorption spectrum of the two dyes eluted from a slide
in normal and pathological specimens without considering the
prepared from a cirrhotic liver. The section was stained as described
in Figure 1 and the color eluted with NaOH-methanol as described relative proportions of collagen types present.
in Figure 2. Once the color equivalences were established, multiple sec-
tions of the five tissues were analyzed for collagen and protein
concentrations using the dye-binding method and the chemical
and was also similar for the five tissues investigated (37.8 procedure. In order to calculate the concentration of collagen
2.05) (Table 2). When the color equivalences of multiple ex- we followed the steps described below:
periments performed with different sections obtained from
1. Determine the absorbances at 605 and 540 nm.
different animals were compared, the values obtained were
2. Calculate the value corresponding to 29. 1% of the op-
38.41 3.27 for Sirius red F3BA and 2.08 0.20 for Fast
tical density at 605 nm, this is the factor that represents
green FCF. From these results, i.e., the presence ofvery similar
the contribution of Fast green FCF to the absorbance
color equivalences for tissues known to contain variable amounts
at 540 nm. This interference is due to the presence of
of interstitial and basement membrane collagens, as well as
a shoulder at that particular wavelength (see Figure 3).
from the histologic observations, most importantly that inter-
3. Subtract the above value from the absorbance at 540
stitial and basement membrane collagens absorb the Sirius red
nm (corrected absorbance).
4. Divide the absorbance at 605 nm and the corrected
Table 1 . Color equivalance of noncollagenous protein as absorbance at 540 nm by their respective color equiv-
determinedfrom the binding of Fast green alences (2.08 and 38.4) in order to obtain the net amount
Absorbance Protein content Color of collagen and noncollagenous proteins in the section.
Tissue (605 nm) (pg/section) equivalence Total protein will then be the sum of both values and
the amount of collagen per mg of protein can be esti-
Liver 0.740 0.066 355.0 2.06 0.118
mated by a simple rule of thumb.
Kidney 0.350 0.093 176.5 1.96 0.132
Lung 0.221 0.002 99.0 2.22 0.025 As illustrated in Table 3, the collagen values estimated by
Heart 0.408 0.005 229.0 1.78 0.021 the dye-binding technique as well as by the chemical method
Spleen 0.185 0.001 83.7 2.21 0.120
are very similar and the range ofdata small, since the individual
standard deviations are also small.
Average 2.04 0.180
In order to further check the reliability of the color equiv-
Mean absorbance ( SD) of 5 to 10 sections obtained from a single animal. alence obtained for collagen, acid-extracted native rat tail ten-
These sections were then used for protein determination by micro-Kjeldahl
don collagen (mainly type I) was dialyzed against 0. 1 M phos-
analysis.
Protein concentration was determined by micro-Kjeldahl analysis of groups phate buffer, pH 7.4, containing 0.2 M NaC1. When the pH
of 3 to 4 sections obtained after elution of the color with NaOH-methanol. of the collagen solution was similar to that of the buffer ( 19),
Values are means.
aliquots containing known concentrations ofprotein were gelled
The color equivalence is expressed as optical density (OD) at 605 nm/mg
of protein. Values are means SD. in multititer wells after incubation at 37#{176}C
for 30 mm. Samples
742 LOPEZ-DE LEON, ROJKIND

Table 3. Collagen concentration in five rat tissues as differences in color equivalence found with native or de-
determined by the chemical and dye-binding methods natured collagen in solution ( 1 , 1 5 ) as compared with the value

Dye-binding procedure
obtained with native fibrillar collagen and formalin-fixed tissue
Chemical method (sag collagen/mg collagen (this article) ( 10 versus 38.4) indicate that other fac-
Tissue (sg collagen/mg protein) protein)5 tors, perhaps the tridimensional organization of the fibrils are
also important.
Liver 5.8 1.1 5.8 0.2
Spleen 17.4 3.3 17.4 1.4
In a previous study we showed that Sirius red F3BA stained
Lung 65.0 14.0 59.1 4.3 equally well interstitial, endothelial, and basement membrane
Kidney 18.9 8.3 17.9 3.7 collagens that had been solubilized with pepsin and their chains
Heart 17.0 4.2 16.8 1.9 separated by SDS-acrylamide gel electrophoresis (1). In this
article we confirmed this observation and we further estab-
Values are means SD of duplicate analysis of tissues prepared from 4
different animals. lished that all collagens, within experimental error, appear to
Values are means SD of at least 10 sections obtained from 4 different bind a similar number of dye molecules. This was inferred
animals.
from the histologic observations that all collagens were stained
red and from the similarities in color equivalences obtained
( 10%) with tissues containing different amounts of collagen
were then stained with 0.2 ml ofa 0.1% solution ofSirius red types. Furthermore, we recently determined the color equiv-
F3BA in saturated picric acid and destained as described under alences for normal and cirrhotic human liver and we obtained
Methods. The values obtained by the chemical and dye-bind- very similar results (A L#{243}pez-De Leon,J Aguirre, and M Rojk-
ing procedures were nearly identical (see Table 4). md, unpublished results).
The method described in this article, in addition to showing
the collagen fibrils and the noncollagenous components very
Discussion neatly, is simple, easy to perform, relatively fast, and allows
Sirius red F3BA is an azo dye with a molecular weight of 1372 several samples to be processed simultaneously. The colors
that has been widely used for the selective staining of collagen can be eluted and the amount of collagen and protein can be
in tissue sections (8,10,1 1,12,20,25,28) and for quantitation established. The method is nondisruptive, and a single section
of collagen that had been solubilized from tissues (9). The dye can be stained several times-if necessary-with great
has four chromophore azo groups and therefore has a very reproducibility.
high extinction coefficient that facilitates the detection of small The staining and destaining conditions described work per-
amounts of protein. As shown in this study, we can detect as fectly well for all the tissues tested, providing that the sectional
little as 5.76 0.2 ,tag of collagen/mg of protein, a value that area and the thickness of the section are within the values
is equivalent to 1 mg of collagen per g of fresh rat liver (24). described. For thick sections or samples with a larger surface
The number of dye molecules that bind to collagens I, II, and area, the amount of reagent to be added and the staining time
III is very constant and has been estimated to be approximately will have to be adjusted. Furthermore, if the amount of col-
126 (9). Although the exact mechanism ofinteraction of Sirius lagen is very high, as it happens in the cirrhotic human liver
red F3BA with collagen is not known, it has been suggested (up to I 50 .tg/mg of protein), the color has to be eluted with
that the sulfonic acid groups of the dye interact strongly with 2 or 3 ml of NaOH-methanol instead of 1 ml. However,
positively charged groups of the protein (9). However, the irrespective of the size and thickness of the section, once the
conditions have been adjusted, the method is quantitative and
reproducible. However, because the exact nature of the in-
teractions between the dyes and the proteins are not known,
Table 4. Protein determination by the chemical and
dye-binding methods of native type I collagen gelled at 37#{176}C and also because we showed staining of the nuclei in some
tissues, it is advisable to always check the section under the
Collagen concentration microscope when standardizing the conditions.
Chemical method Dye-binding method5
Although this method solves the problem of quantitation
(pg) (sag) of collagen in small samples, an equally important problem
still remains; this is related to the sampling error that occurs
10 12.7
when a small needle is introduced into a 500-1500 g liver
12.2
that contains a heterogeneous lesion, i.e., a macronodular cir-
30 33.3
rhosis. Work is in progress to establish the minimum size of
35.4
40 41.6 human liver required to assess quantitatively and accurately
43.7 the amount of collagen in a biopsy.
50 46.3
5 1.5
Acknowledgments
The authors wish to acknowledge the secretarial assistance of Ms. Josefina
Hydroxyproline analysis of collagen was performed prior to formation of
Quiroga. They are indebted to Mrs. Alicia B:fanoforpreparing the tissue
the gel.
Gelled samples were stained with Sirius red as described under Methods. section and to Dr. Victor Tsutsumi for his help in mounting the photo-
Duplicate experiments. graphic material.
MICROMETHOD FOR COLLAGEN DETERMINATION IN TISSUE SECTIONS 743

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