Document dnZ92Dey49j150ky9pL7LqXo5
0022-1554/83/33.00
The Journal of Histochemistry and Cytochemistry
Copyright 1983 by The Histochemical
Society,
Inc.
Vol. 31, No. 10, pp. 1183-1189,
1983
Printed in U.S.A.
II
Quantitative
Immunohistochemistry:
A Comparison of Microdensitometric
Analysis of Unlabeled
Antibody Peroxidase-Antiperoxidase
Staining and of
Microfluorometric
Analysis of Indirect Fluorescent
Antibody
Staining for Nicotinamide
Adenosine Dinucleotide
Phosphate
(NADPH)-Cytochrome
c (P-.450) Reductase in Rat Liver1
MARTYN
T. SMITH,2
JAN A. REDICK,
and JEFFREY
BARON3
Toxicology Unit, Department England and The Toxicology City, Iowa 52242
ofPharmacolog.y
(M.T.S.). The School ofPharmacy,
Center, Department
of Pharmacology
(J.A.R.;J.B.),
University ofLondon.
London WCJN lAX.
College of Medicine, The University of Ioua, Iowa
Received for publication August 24, 1982 and in revised form February 9, 1983; accepted February 19, 1983 (OA 82-321)
The intralobular
distribution
of nicotinamide
adenine di-
nucleotide phosphate (NADPH)-cytochrome
c (P-450) re-
ductase (NADPH:ferricytochrome
oxidoreductase,
EC
1.6.2.4) in rat liver has been investigated
by means of two
quantitative
immunohistochemical
techniques: microden-
sitometric quantitation
of unlabeled antibody peroxidase-
antiperoxidase
staining
and microfluorometric
analysis of
indirect fluorescent
antibody
staining.
Utilizing
sheep
antiserum elicited against NADPH-cytochrome
c (P-450)
reductase that had been isolated and purified to apparent
homogeneity
from rat liver microsomes, the reductase was
detected within hepatocytes
throughout
the liver. How-
ever, differences in the intensity of staining of hepatocytes
within different regions of the liver lobule were readily
apparent after completion
of both immunohistochemical
staining procedures.
These visual findings were verified
by microdensitometric immunohistochemical
and microfluorometric
analyses of
staining, both of which revealed that
approximately
the same degree of staining for NADPH-
cytochrome
c (P-450) reductase was produced within the
centrilobular
and midzonal regions of the liver lobule,
whereas periportal
hepatocytes
were stained with signif-
icantly less intensity. These results demonstrate
that the
application ofeither microdensitometry
in conjunction with
unlabeled antibody peroxidase-antiperoxidase
staining or
microfluorometry
after indirect fluorescent antibody stain-
ing can be used to quantitatively
determine the intratissue
distributions
KEY WORDS:
of antigens. NADPH-cytochrome
c(P-450) reductase; Rat liver;
Immunohistochemical
localization;
Quantitative
immunohis-
tochemistry;
Microdensitometry;
Microfluorometry.
Introduction
Immunohistochemical
techniques
are now widely used to de-
termine the localizations
of enzymes and other substances of
biological
importance
within tissues and cells. Studies em-
ploying these techniques,
however, are usually of a qualitative
nature, with little or no attempt being made to obtain quan-
`Supported by a grant from the Medical Research Council of Great Britain and by United States Public Health Service Grants GM 12675 and CA 30140.
2Present address: Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720.
1'o whom reprint requests should be addressed.
titative data. Since the ability to quantitate
cellular antigens
would provide a great deal of important biological information,
methods for the quantitation
ofimmunohistochemical
findings
should be sought.
This laboratory has employed both unlabeled antibody per-
oxidase-antiperoxidase
and indirect fluorescent antibody staining
techniques
to investigate
the intrahepatic
localizations
and dis-
tnibutions of several enzymes, including nicotinamide
adenine
dinucleotide
phosphate
(NADPH)-cytochrome
c (P-450) re-
ductase (1,10,16,17),
cytochromes
P-450 (2,3,10), and epox-
ide hydrolase (7,10), that participate
in the bioactivation
and
detoxification
of endogenous
and exogenous
substances.
In
these studies, microfluorometric
determinations
of the inten-
sities of fluorescence
emitted from hepatocytes
after indirect
fluorescent
antibody staining provided semiquantitative
data
I 183
1184 SMITH, REDICK, BARON
regarding the relative extents of specific antibody binding within
centnilobular,
midzonal,
and peniportal
regions of the liver
lobule.
In the present study, scanning and integrating
microden-
sitometry was employed
to obtain quantitative
data from liver
sections stained for NADPH-cytochrome
c (P-450) reductase
utilizing the unlabeled
antibody
peroxidase-antiperoxidase
staining method. Results of microdensitometnic
analyses were
directly compared
to microfluorometnic
measurements
of in-
direct fluorescent
antibody staining for the reductase.
In ad-
dition, microdensitometnic
measurements
within individual
hepatocytes
allowed for the determination
of the exact intra-
lobular distribution
of NADPH-cytochrome
c (P-450) reduc-
tase within rat liver.
Materials
and Methods
Purification
of NADPH-cytochrome
c (P-450) reductase
and
production
of antibody.
NADPH-cytochrome
c(P-450) reductase,
solubilized by tryptic digestion of rat liver microsomes, was purified
to apparent homogeneity employing minor modifications (18) of the
method described by Omura and Takesue (9). Sheep antiserum to the
reductase was obtained as described previously ( 18), and whole sheep
anti-reductase
and normal (nonimmune)
sera were employed
in the
immunohistochemical
staining procedures.
Normal sheep serum, the
soluble sheep peroxidase-antiperoxidase
complex, rabbit antiserum
to sheep immunoglobulin
(Ig) G, and fluorescein isothiocyanate (FITC)
conjugates of IgG prepared from rabbit antiserum to sheep IgG were
obtained from Miles Laboratories,
Inc.
Immunohistochemical
staining
procedures.
Male albino
Holtzmas4 rats, weighing 180-230 g, were used. They were allowed
food and water ad libitum. Rats were killed by decapitation, the livers
were immediately excised, and the median lobe of each was cut into
blocks approximately
2 mm in thickness. The liver blocks were fixed
at 4#{176f}oCr a total period of 4 hr by immersion in several changes of
a solution containing 0.35% (w/v) parabenzoquinone
(Polysciences,
Inc.) and 0.02 M CaCl2 in 0.2 M sodium cacodylate buffer, pH 7.4.
The fixed blocks were then dehydrated, cleared, embedded in paraffin,
and serial sections 7 .tm in thickness
were prepared,
placed on al-
bumin-coated
slides, and dried overnight
at 37#{176}CP. rior to perfor-
mance of the immunohistochemical
staining procedures, the sections
were dewaxed in xylene and rehydrated in graded methanols.
The immunohistochemical
localization of NADPH-cytochrome
c
(P-450) reductase in rat liver was accomplished
employing
minor mod-
ifications of the unlabeled antibody peroxidase-antiperoxidase
(1-
3,7,15-18)
and the indirect fluorescent
antibody (2,3,7,16-18)
stain-
ing techniques. In the unlabeled antibody peroxidase-antiperoxidase
method, after the endogenous peroxidase activity of the tissue had
been blocked ( 1), the sections were exposed for 40 mm to 10% (v/
v) dimethyl sulfoxide prior to completion of the staining protocol ( 1).
We have found that dimethyl sulfoxide enhances the uniform pene-
tration of antibodies into tissue sections and yields much more re-
producible immunohistochemical
staining. In the indirect fluorescent
antibody technique,
the sections were similarly exposed to dimethyl
sulfoxide, and after exposure to either sheep anti-reductase
serum or
normal sheep serum (both ofwhich had been diluted 1:500 with 0.05
M Tnis-HC1 buffer, pH 7.75, containing 0.154 M NaC1), they were
exposed for 1 hr at 37#{176t}oC FITC-conjugated
IgG of rabbit antiserum
to sheep IgG that had been diluted 1:100 with Tnis-buffered saline.
The sections were then examined by incident-light fluorescence mi-
croscopy using a modified Leitz Orthoplan microscope ( 19) with a
1 50-W Osram xenon lamp and a Leitz Ploemopak
2. 1 fluorescence
illuminator
containing
a K2 filter block (excitation,
470-490
nm; RKP
5 10 beam-splitting
mirror; LP 5 1 5 suppression
filter) (2).
Microfluorometric immunohistochemical
and microdensitometric staining. The fluorescence
quantitation
of
emitted from 2.5
x 2.5 j.m areas within hepatocytes
was transmitted
through a Leitz
microfluorometric
attachment
to a Schoeffel GM 100 monochroma-
tor, and fluorescence emission at 525 nm was detected by an EMI
9658A photomultiplier
tube. The signal from the photomultiplier
tube
was then amplified by a Schoeffel M 460 photometer. Because fluo-
rescence intensity should be linearly related to the concentrations
of
FITC and antigen, and because absorbance is a linear function of
sample concentrations,
whereas transmittance
is a logarithmic
func-
tion, the output from the photometer
was fed into a log-linear
con-
verter designed and built by the Bioengineering
Facility of The Uni-
versity of Iowa College of Medicine
that converts the fluorescence
emission intensity (transmitted
light) into an absorbance
value. The
output from the log-linear
converter
was then displayed on an Axiom
EX-801P digital printer (Axiom Corp.). Since absorbance
decreases
as the intensity was subtracted
ofemitted
fluorescence
increases, the absorbance
value
,from 1 and the results are expressed
in terms of
1 - absorbance ( x 100). In this manner, a positive, linear relationship
is obtained between the intensities
of indirect fluorescent
antibody
staining and the microfluorometric
measurements.
A uranyl glass stan-
dard attached to a Plezy adaptor was employed
to calibrate the mi-
crofluorometric
apparatus.
By doing this, less than 5% variability
is
found in microfluorometric
measurements
obtained
from within a
given section and from within corresponding
hepatocytes
in multiple
liver sections.
Osmium black, the product formed by the osmication
of oxidized
3,3'-diaminobenzidine
in the unlabeled antibody peroxidase-antiper-
oxidase staining technique
(1 1), was quantitated
using a Vickers M85
scanning and integrating
microdensitometer.
The use of this instru-
ment to overcome problems associated with the measurement of het-
erogeneously
distributed
reaction products in tissue sections has been
discussed in detail by Chayen (4). The microdensitometer
was inter-
faced to a CBM PET microcomputer
to facilitate data handling and
statistical analysis (14). As seen from the data presented
,in Figure 1 osmium
black exhibits
a relatively broad absorption band having a maximum at 430 nm. The
absorbance of osmium black at 430 nm was determined in tissue
sections using a X 40 objective,
spot size 1 (being 0.5 .tm in diameter
in the optical plane of the specimen),
and a field size of 5 m diameter.
Using these conditions,
it was possible to obtain microdensitometric
measurements
from within the cytoplasm
of individual
hepatocytes.
The results of microdensitometric
analyses of unlabeled antibody per-
oxidase-antiperoxidase
staining are expressed in terms of integrated
absorbance units. The Vickers microdensitometer
was calibrated be-
fore use by employing a series of filters of known absolute absorbance
(4).
For quantitative analyses of immunohistochemical
staining for
NADPH-cytochrome
c (P-450) reductase
within liver, two pairs of
serial sections were prepared from each of 6 livers. One section of
each pair was exposed to sheep anti-reductase section was exposed to normal sheep serum.
serum, and the other One pair of sections was
then stained employing the indirect fluorescent antibody method, while
the second pair was stained using the unlabeled antibody peroxidase-
antiperoxidase
technique.
In each tissue section, microfluorometnic
or microdensitometric
measurements were taken from 1 5 hepatocytes
lying within the first 5 cell layers surrounding
central veins of less
than 40 am diameter (centrilobular
hepatocytes),
from 1 5 hepatocytes
lying within 3 cell layers on either side of midlines between central
veins and portal tracts (midzonal
hepatocytes),
and from 1 5 hepato-
cytes lying within the first 5 cell layers surrounding portal tracts of
HEPATIC
NADPH-CYCTOCHROME
c (P-450) REDUCTASE
1185
500 C 400
0W-.
0 300
SD 200
.
100
0 400 600 700
Wavelength (nm)
Figure 1 . Absorption
spectrum
of osmium black determined
in sec-
tions of rat liver by means of scanning and integrating
microdensi-
tometry. The vertical dashed line is at 430 nm.
less than 40 am diameter
(peniportal
hepatocytes).
To calculate the
fluorescence
or absorbance
due to anti-reductase
binding to centri-
lobular, midzonal,
and periportal
hepatocytes,
the mean microfluo-
rometric or microdensitometric
value obtained from within each re-
gion in the section exposed to normal sheep serum was subtracted
from each individual
measurement
obtained from within the corre-
sponding
region in the serial sections exposed to sheep antiserum
to
NADPH-cytochrome
c (P-450) reductase. Mean SEM values re-
ported in Tables 1 and 2 were calculated
from the values obtained
from each liver.
Statistical analysis. The results of microdensitometric
analyses
were compared
to those obtained by microfluorometry
by estimating
the pooled variance and performing one-way analyses of variance on
the data obtained with each method. Where appropriate,
the group
Student's t-test was also used. The level of significance
was taken as
5% (p < 0.05). The coefficient
ofvariation
ofthe data for each region
was calculated for both methods in order to compare the relative
variability
of the data produced.
The use of the coefficient
of variation
as an indicator of the degree of variability eliminates the need to take
into account the fact that the microdensitometnic
values for anti-re-
ductase binding were numerically
greater than the microfluorometric
values.
Results
Immunohistochemical
Cytochrome
c (P-450)
Localization Reductase
of NADPHin Rat Liver
As reported previously
by this laboratory
(1,10,16,17),
when
sections of rat liver were exposed to sheep antiserum to rat
hepatic microsomal
NADPH-cytochrome
c (P-450) reductase
in both the unlabeled
antibody
peroxidase-antiperoxidase
and
the indirect fluorescent
antibody staining protocols,
hepato-
cytes throughout
the liver were stained for the enzyme
(Figure
2A,C). Immunohistochemical
staining for the reductase
was
not apparent
within hepatocytes
in sections that had been
exposed to normal sheep serum (Figure 2B,D). It is readily
apparent from the photomicrographs
in panels A and C of
Figure 2, however, that peniportal hepatocytes
are much less
intensely stained for the reductase than are either centnilobular
or midzonal hepatocytes.
This observation
indicates that pen-
portal hepatocytes
contain less enzyme than do those cells
lying within the centrilobular
and midzonal
regions of the liver
lobule. To quantitatively
investigate
this possibility,
micro-
fluorometnic
and microdensitometnic
analyses were conducted
to determine
the extent of anti-reductase
binding to hepato-
cytes lying within the three regions of the liver lobule.
Microfluorometric
Quantitation
Fluorescent
Antibody
Staining
of Indirect
The results of microfluorometnic
analyses of indirect fluores-
cent antibody staining of centnilobulan,
portal hepatocytes
are shown in Table
midzonal, and pen1 . The fluorescence
emitted from within cells in sections exposed to normal sheep
serum is due to both nonspecific
tissue autofluorescence
and
nonspecific
binding of FITC-conjugated
IgG. In all instances,
cells in sections exposed to sheep anti-reductase
serum emit
fluorescence
with significantly
(p < 0.01) greater intensity. In
an earlier study (16), removal of the antibody from the anti-
serum by adsorption
with purified rat hepatic microsomal
NADPH-cytochrome
c (P-450) reductase was shown to cause
the intensity of emitted fluorescence
to decrease to that mea-
sured within cells in sections exposed to normal sheep serum.
The data summarized
in Table 1 indicate that the anti-reduc-
tase bound equally to centnilobular
and midzonal hepatocytes,
while significantly
less anti-reductase
bound to peniportal he-
patocytes. These microfluorometnic
findings are thus consist-
ent with visual observations
of indirect fluorescent
antibody
staining for the reductase within the liver lobule (Figure 1C)
and are also in agreement
with those reported
previously
(10,16,17).
Microdensitometric
Quantitation
Antibody
Peroxidase-Antiperoxidase
of Unlabeled Staining
The amount of osmium black deposited
within the cytoplasm
of centrilobular,
midzonal,
and peniportal
hepatocytes
after
unlabeled
antibody
peroxidase-antiperoxidase
staining was
determined
microdensitometnically
(Table 2). The mean ab-
sorbance of sections that had not been subjected to staining
was approximately
0. 1 3 within all regions of the lobule (data
not shown), whereas sections exposed to normal sheep serum
in the staining protocol had an overall mean absorbance
of
approximately
0.28 (Table 2). The absorbance
values deter-
mined after exposure
to normal sheep serum, therefore,
rep-
resent the amount of absorption
due to both light scattering
and nonspecific
staining. As seen from the data presented
in
Table 2, the absorbance
of cells in sections exposed to sheep
anti-reductase
serum was in all cases significantly
(P < 0.01)
greater than that of cells in sections exposed to normal sheep
serum.
Calculation
of the absorbance
due to anti-reductase
binding revealed that the antibody bound to similar extents
within centnilobular
and midzonal regions of the liver lobule,
whereas significantly
less binding occurred within peniportal
regions. After the anti-reductase
had been removed from the
antiserum
by adsorption
with the purified enzyme, anti-re-
ductase binding was decreased
by approximately
90%: the
1186
Figure 2. Immunohistochemical
lo-
calization
of NADPH-cytochrome
c
(P-450) reductase
within rat liver. The
photomicrographs
show areas in
thick sections prepared
from the liver
of a male rat. (A) Section exposed to
sheep anti-reductase
serum in the un-
labeled antibody
peroxidase-antiper-
oxidase staining protocol.
(B) Serial
section exposed to normal sheep serum.
(C) Section exposed to sheep anti-re-
ductase serum in the indirect fluores-
cent antibody
staining protocol.
(D)
Serial section exposed to normal sheep
serum. The sheep anti-reductase
serum
and normal serum had each been di-
luted 1:500 with 0.05 M Tnis-HCI
buffer, pH 7.75, containing 0.154 M
NaCI. Central veins (V) and portal tracts
(P) are indicated in B and D. Original
magnification
x 165. Bar = 200 j.em.
SMITH, REDICK, BARON
V V
p
mean SEM absorbance
at 430 nm of centnilobular,
mid-
zonal, and periportal
hepatocytes
in sections exposed to ad-
sorbed anti-reductase
serum was 0.37 0.02, 0.34 0.02,
and 0.31 0.02, respectively.
These microdensitometnic
find-
ings are thus in agreement
with those obtained from micro-
fluorometric
analyses and further demonstrate
that NADPH-
cytochrome
c (P-450)
reductase
is not uniformly
distributed
within the liver lobule.
Comparison
of Microftuorometric
Microdensitometric
Determinations
and
Results of microdensitometnic peroxidase-antiperoxidase
analyses of unlabeled antibody staining for N ADPH-cytochrome
C (P-450)
reductase
within the liver lobule were directly
com-
pared to those obtained
by microfluorometry
following
mdi-
rect fluorescent
antibody
staining
by means of one-way
anal-
yses of variance.
The results of these analyses
demonstrated
that there was no significant
difference
(p > 0.05) in the extent
of anti-reductase
binding
to centnilobular
and to midzonal
he-
patocytes
as determined
by microfluorometry
and microden-
sitometry, whereas both methods showed that significantly less
(p < 0.001)
anti-reductase
bound to peniportal
hepatocytes
(Tables 1 and 2). Thus, the two methods
for the quantitation
of immunohistochemical
staining
yield very similar
The coefficients
of variation of the values determined
results. with the
two methods for the extents of antireductase
binding within
centnilobular,
midzonal,
and peniportal
regions
of the liver
HEPATIC
NADPH-CYCTOCHROME
c (P-450) REDUCTASE
1187
Table 1 . Microftuorometric measurements of the intensity of indirect fluorescent antibody staining for NADPH-cytochrome reductase within different regions of the liver lobule"
c (P-450)
Region
Emitted fluorescence after exposure to sheep antireductase serum
Emitted fluorescence
after
exposure to normal
sheep serum
Fluorescence
due to
anti-reductase
binding
Centnilobular
69.1 0.3
Midzonal Periportal
68.3 0.4 55.7 0.4
`The values are given as the mean S.E.M. of six rats and are expressed in terms
bValues are not significantly different from each other, p > 0.05.
`Value is significantly lower than corresponding
values from the centrilobular and
43.9 43.4 43.7 of 1 - absorbance
midzonal regions,
0.2 0.3 0.5
( X 100).
p < 0.001.
25.2
24.9 0,6b
12.0 0.5'
lobule were found to be 0.038, 0.058, and 0.092, respectively,
by microfluorometry
and 0.145, 0.120, and 0.144, respec-
tively, by microdensitometry.
From these findings,
it can be
concluded
that there was an average
of 2 to 3 times more
variation
in the microdensitometnic
values than in the micro-
fluorometric
values. This may, in part, reflect the potentially
greater accuracy of microfluorometry
over microdensitometry
(8).
Determination
of the Intralobular
Curve of NADPH-Cytochrome
in Rat Liver by Microdensitometry
Distribution c (P-450) Reductase
The intralobular
distribution
of the reductase was further in-
vestigated
by microdensitometnically
determining
the absor-
bance at 4 30 nm from within 1 5 to 20 hepatocytes
situated
along at least four imaginary
straight
lines between
selected
central veins and portal tracts in each tissue section. Subtrac-
tion of the absorbance
due to light scattering and nonspecific
staining, that is, values from sections exposed to normal serum,
from the absorbance
measured within corresponding
cells in
serial sections exposed to anti-reductase
serum generated
the
intralobular
distribution
curve for the binding of the anti-
reductase within the liver lobule. By pooling the values ob-
tamed from 4 rats, it was possible to produce the mean intra-
lobular distribution
curve of NADPH-cytochrome
c (P-450)
reductase in rat liver shown in Figure 3. From these data, it
can be seen that hepatocytes
adjacent to the central vein con-
tam approximately
twice as much reductase
as do those ad-
jacent to the portal tract. While the content of the reductase
was found to be fairly constant within both centnilobular
and
periportal hepatocytes,
variability in enzyme content was noted
among midzonal cells, especially when the content within those
cells adjacent to the centnilobular
region was compared to that
within midzonal cells that are adjacent to the peniportal region.
Discussion
We previously
reported (1,10,16,17)
that an inhibitory anti-
body directed against rat hepatic microsomal
NADPH-cy-
tochrome c (P-450) reductase could be used to determine
both
the cellular localization
and the intralobular
distribution
of the
enzyme within rat liver. In these investigations,
visual obser-
vations of unlabeled antibody peroxidase-antiperoxidase
and
indirect fluorescent
antibody staining revealed that, although
present within all hepatic parenchymal
cells, NADPH-cyto-
chrome c (P-450) reductase was not uniformly distributed across
the liver lobule. Microfluorometric
measurements
of the in-
tensity of indirect fluorescent
antibody staining provided semi-
quantitative
data, in terms of relative fluorescence
units, for
similarities
and differences
in the extents of anti-reductase
binding to hepatocytes
within the different regions of the liver
lobule.
Quantitative
data has now been obtained from sections of
rat liver stained for NADPH-cytochrome
c (P-450) reductase
using the unlabeled antibody peroxidase-antiperoxidase
method.
In this immunohistochemical
staining technique,
after the anti-
reductase has interacted with and bound to the enzyme present
in the tissue section, it is coupled to a peroxidase-antiper-
oxidase complex. The subsequent
exposure of this complex
to 3,3'-diaminobenzidine
and H202 results in the precipitation
of oxidized diaminobenzidine
that, when chelated with 0504,
Table 2. Microdensitometric
measurements of the intensity of unlabeled antibody peroxidase-antiperoxidase
NADPH-cytochrome
c (P-450) reductase within different regions of the liver lobule"
Region
Absorbance
after exposure
to sheep anti-
reductase serum
Absorbance
after exposure
to normal sheep serum
Centnilobular Midzonal Peniportal
81.9 1.1 75.7 1.8 57.6 1.6
30.0 2.6 28.0 2.2 26.0 1.5
`The values are given as the mean SEM of six rats and are expressed as integrated
absorbance
units ( X 100).
bValues are not significantly different from each other, p > 0.05.
`Value is significantly lower than corresponding
values from the centrilobular
and midzonal regions, p < 0.001.
staining for
Absorbance
due to
anti-reductase
binding
51.9 3.1' 47.7 2.3' 31.6 1.9'
1188 SMITH, REDICK, BARON
I w
(1)
.,
8
*
;
C I.
F
4 :, :
I,
a
centri lobular region
I mid-zone
periprt region
1 3 5 7 9 11 13 15 17
Distance from Central Vein
( N#{1o7f6} Hepatocytes)
Figure 3. Intralobular distribution curve of NADPH-cytochrome
(P-450) reductase in rat liver determined by microdensitometry
unlabeled antibody peroxidase-antiperoxidase
staining. The
are given as the mean SEM of at least 16 determinations
using sections of livers prepared from 4 rats.
c after values made
less staining
occurred
within the periportal
regions.
Moreover,
a high degree of statistical
correlation
was found between
the
results obtained
with the
From these observations, tent ofNADPH-cytochrome
two quantitative
methods.
it can be concluded
c (P-450)
reductase
that the conis 1.6 to 2.0
times greater
in centrilobular
hepatocytes
than in periportal
hepatocytes.
This distribution
is, thus, very similar to that of
hepatic cytochrome
P-450 (5,12). It should be borne in mind
that the quantitative
immunohistochemical
techniques
used in
this study reveal only the presence
of the enzyme
and not its
activity. It is very likely, however,
that the activity of NADPH-
cytochrome
c (P-450)
reductase
is greater
in the centrilobular
region than in the periportal
region of the lobule, since it has
recently been shown that centrilobular
hepatocytes
have far
more NADPH
available for monooxygenation
reactions ( 13)
and metabolize
7-ethoxycoumarin
at a faster rate (6) than do
those hepatocytes
situated in the periportal region. It is, there-
fore, highly probable that centrilobular
hepatocytes
are more
important
in the Phase I metabolism
of a majority
of drugs
and other foreign compounds
than are periportal hepatocytes.
Acknowledgments
The authors wish to thank Professor helpful discussion and NP. Jewell, help with the statistical analyses.
ED. Willsfor his encouragement M. Francis, and]. Schulman for
and their
produces
a brown particulate
deposit (osmium black) at the
site of the antigen-antibody
complex ( 1 1 ). In the present in-
vestigation,
the amount of osmium black formed within he-
patocytes
was quantitated
using a scanning and integrating
microdensitometer.
The microdensitometric
results were then
directly
compared
to those obtained
by microfluorometric
quantitation
of indirect fluorescent
antibody staining for the
reductase within the three regions of the liver lobule.
Although
microfluorometnic
quantitation
is theoretically
more precise than microdensitometry
(8), microdensitometric
quantitation
of unlabeled antibody peroxidase-antiperoxidase
staining has a number of distinct advantages
to offer over the
microfluorometric
quantitation
of indirect fluorescent
anti-
body staining. First of all, the unlabeled antibody peroxidase-
antiperoxidase
method produces a superior resolution
of in-
tracellular
staining than does the indirect fluorescent
antibody
staining method. Secondly, peroxidase-antiperoxidase
stain-
ing is stable, whereas
fluorescent
staining
is not and, thus, is
not subject to errors such as those resulting from the fading
of the stain. Finally, microdensitometric
measurements
can be
expressed
as absolute units of absorbance,
so that if the molar
absorptivity
of the reaction product is known, the values ob-
tamed can be converted
into conventional
biochemical
units.
Statistical analysis ofthe results obtained in this quantitative
immunohistochemical
investigation
revealed that microden-
sitometric
measurements
of unlabeled
antibody peroxidase-
antiperoxidase
staining and microfluorometnic
quantitation
of
indirect
fluorescent
antibody
staining for NADPH-cyto-
chrome c (P-450) reductase yielded very similar results. Both
methods showed that approximately
the same degree of stain-
ing for the reductase
was produced
within the centrilobular
and midzonal regions of the liver lobule, whereas significantly
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