0022-1514/90/13.30
Vol. 38. No. 12, pp. 1801-1814. 1990
Printed in US.A.

The Journal of Histochemistry and Cytochemistry
Copyright 0 1990 by The Histochemical Society, Inc.

Original Article

1
Light Microscopic Autoradiography for Study of Early
Changes in the Distribution of Water-soluble Materials'
TADAFUMI KAWAMOTO
Department of Biochemistry, School of Dental Medicine, Tsurumi University, Tsurumi, Yokohama,Japan.
Received for publication February 27, 1990 and in revised form July 11, 1990; accepted July 20, 1990 (OA1918).

An approach using autoradiography for the study of earlychanges in the distribution of water-soluble materials andthe chemograph involved was investigated. Radioactive calcium chloride (* Ca) was injected into the iliac vein of a rat.
Ten seconds after the injection the rat was frozen in hexane(-9O'C). The frozen rat was embedded in 5% sodium carboxymethyl cellulose and blocked in the coolant. A sheetof plastic tape coated with a synthetic rubber glue was
fastened to the trimmed block surface, and whole-body sections 2-10 pm thick were cut with a disposable microtome
knife. Selected sections were freeze-dried and then covered
with a dried autoradiographic emulsion film about 1 pm
thick. The autoradiograph clearly showed the distribution

Y

Introduction
Autoradiography has been widely utilized to study the distribution of drugs and metabolites in the body. The procedure can beused to examine the distribution of both insoluble materials andsoluble materials. The conventional histological procedure, consisting of fixation, graded solvent dehydration, and embedding,can be employed for the former purpose but is not suitable for thelatter because the soluble tracer is lost by diffusion from its original site. In consequence, a number of techniques have been evolved
to avoid the diffusion of soluble tracer during the preparation of
specimens (5,6,9,11,12,22,24,27).In most cases, after the administration of a tracer, tissues were dissected from the animal and freezesectioned, and the sections were used for the light microscopic autoradiography. However, because the distribution pattern of tracers
may change during dissection of the tissue, the only way to avoid
the problem is to use a whole-body sectioning technique accompanied by direct dry mounting of a photographic emulsion film.
However,the autoradiograph prepared by thisprocedure often yields
false results caused by a chemical reaction between the specimen
and the photographic emulsion (a process known as chemography).
In the present study, an attempt was made to prevent, as far as
possible, the migration of diffusible radioactive substances and to'

Supported in part by Research Grant No. 62771509 from the Ministry of Education of Japan.

of radioactive calcium in the calcification zone of long bones.
The samples chosen to assess chemographic artifacts showed
positive and negative chemographies on most of the tissues
when these were kept at 23'C, and although both chemographic effects were significantly reduced when the samples
were kept at -20°C, cells in several tissues still exhibited
positive and negative chemographies. The technique can beused for the study of any animal whose size is suitable forwhole-body freeze-sectioning. (1Hisrochem Cycochem
38:1805-1814, 1990)
KEY WORDS:Autoradiography; Soluble material: Chemog-

raphy.

identify chemographic changes occurring in autoradiographs offreeze-dried sections prepared by this method.

Materials and Methods
Preparation of Frozen Sections. In this study, 45Ca was selected as a
typical water-soluble tracer. A 9-dayold rat of the Wistar strain (weighing
about 20 g) was anesthetized with ether. and 5 pCi of 45CaC12/gbody
weight in a volume of 0.1 ml of saline solution was injected into the iliac
vein. After exposure for 10 sec. the animal was rapidly frozen by immersion
in hexane at - 90'C cooled with a cooling apparatus (Ncocool Bath, Model
BD-51; Yamato Scientific CO;Tokyo,Japan). The frozen animal was wipedwith gauze and immersed in a brass container (3 x 4 x 12 Em) filled with
5 % sodium carboxymethyl cellulose solution (CMC). As soon as possible.
the container was put into the coolant and the CMC was completely frozen. The frozen block was then taken out of the container and attached
to a sample holder with CMC. The holder was fastened to the microtome
stage with screws and left in the cryostat ( - 2O'C) (LKB 2258 PMV Rockville, MD) to acclimatize for more than 3 hr before sectioning. The block
was trimmed with a disposable microtome blade (8 un in length; Type CK35;
Feather CO,Gifuken, Japan) attached to the blade holder (Figure 1). The
exposed tissue surface was covered with a specially prepared adhesive plastic tape and placed in close contact with the block surface by rubbing thetape with a cotton wad. The tape was prepared by placing a sheet of polyvinylidene chloride film (Saran Wrap, 5 pm thick) on an acrylic board wetted with 100% ethanol, and then coated with a synthetic rubber glue (Nenchakunori; Azia Gensi Co., Gifuken, Japan) which was diluted by adding
two or three parts of normal hexane to one part of the glue. After drying,
1805

KAWAMOTO

1806

Figure 1. Microtome with a carboxymethyl cellulose block and a disposable
microtome blade installed. Arrow A indicates a blade holder and arrow B indicates a disposable microtome blade.

the prepared film was cut with a rotary cutter (Typc S; Olfa Co. Osaka,
Japan) or surgical knife to an appropriate size. The frozen sections (2-10
pm thick) supported by the adhcsivc tapc were cut with a cutting angleof 30" and a cutting specd of less than 0.5 mlmin. The cut sections wereplaced on a metal plate cooled in thc cryostat, and the section surface wascovrrcd with a sheet of glwinc papcr (Hakuai CO;Tokyo,Japan) and pressedwith a prc-cooled rubber roller to cnsure a close contact between the section and the tape. The papcr was then removed from thc section.
Autoradiography. The corners of the tape which mounted the section
wcrc fixcd on a prc-cooled glass slide with double-sidcd adhcsivc tape in
the cryostat. Sections were then freeze-dried for more than 12 hr, and thcn
placed in a desiccator containing silica gcl within the cryostat to prcvcnt
thc occurrcnce of condensation on the section whcn they wcrc removed
from the cryostat. Aftcr this the desiccator was removed from the cryostat
and allowed to reach room temperature. For examination of thc distribution of tracer in the whole body, sections 3-10 pm thick wcrc sclcctcd andthe section surfacc was then covered with a plastic film (Diafoil, 4 pm thick;Diafoil CO, Tokyo, Japan) to prcvcnt chcmography. A piece of X-ray film
(double-coated film, Fuji Typc 100; Fuji Photo CO,Tokyo,Japan) was placedin contact with the Diafoil covering the section. The spccimcns were thenstored in a light-tight box containing silica gcl and placed in a refrigerator
at 4'C. For subscqucnt development the film was wettcd with water, andthen carefully floatcd on the developer (Rcndol; Fuji Photo CO)for 5 min
at 2O'C (9). After dcvcbpmcnt. the film was submerged in the photographic
fixative (Fuji Fix, Fuji Photo Co.) for 10 min. A serial section adjacentto that uscd for autoradiography was stained with hematoxylin and eosinfor combined examination of the histology and the distribution of tracer,and mounted with glyccrin.
For light microscopic autoradiography, sections 2-5 pm thick were used.
and dried thin films of autoradiographic emulsion used in light microscopic autoradiography wcrc prepared according to the previously reported
method (9). In brief, 0.4 ml of 2% aqueous solution of sodium dioctylsulfosuccinatc was addcd to 5 g of autoradiographic emulsion (Sakura NRM2; Konica Co. Japan) melted at 40'C. and thc solution was mixed by stirring. Aftcr being left for about 1 hr, a small amount of thc cmulsion was
collcctcd by dipping thc tip of a glass tube (1.3 cm in diamctcr) into theemulsion and a bubble was cardully blown to a diamctcr of approximatcly
6 cm. The bubble was allowcd to air-dry until it began to wrinklc. andtwo g l w slidcs with a section mounted on cach were placed so that cachsection side faccd thc bubble. The g l w slidcs wcrc then moved together

until thc bubble burst. This resulted in an emulsion film on cach section,
which was made to adhere closely to the section by breathing moist air on
it. For cxposurc. the emulsion covered samples were stored at -2O'C ina light-tight box containing silica gcl. After exposure the emulsion on thesection was developed for 5 min at 20°C and fixed. After rinsing with water. the section was stained with hematoxylin and eosin. Then the section
mounted on the plastic tapc was trimmed with a surgical knife and scparated from the glass slide. It was thcn rc-mounted between a glass slideand a coverslip with glycerin, or dehydrated with graded ethanol andmounted with cntcllan.
To examine the thickncss of the emulsion film thus produced. after
drying the film was embedded in Epok 812 and placcd in a 60'C oven topolymerize. Ultra-thin sections of thc film wcrc cut with a diamond knifc
using an Ultrotomc (LKB; Ultrotomc V 2088) and then coated with a carbon film by evaporation. The distribution ofsilvcr halide grains in the film
and the thickncss of the film wcrc examined by means of a transmission
clcctron microscope (2000EX; JEOL). The relationship between the radioactivity and the autoradiographic optical density was examined with standard samplcs containing various amounts of 4'Ca. The samples were prepared by the following procedures. Six 5 % gclatin solutions containing
different amounts of radioactivc calcium were prepared. Each solution was
put into small cavities (a diamctcr of 4 mm) made in a frozen carboxymethyl
ccllulose block (CMC) and frozen in hexane (-9O'C). The CMC block was
mounted to the sample holdcr of the cryotomc. Eighteen serial sections
wcrc cut and each section was fixed on the glass slides with double-sided
adhcsive tapc and freeze-dried. Thc sections were subdividcd into thrcc
groups of six sections and were placed in contact with glass slides covered
with one to thrcc layers of emulsion. Two sections from each group werethcn exposed for 2, 3. or 4 days. After exposure the film and thc section
wcrc scparatcd. and the film was developed for 5 min at 20'C and fixed.
The radioactivity of cach sample trimmed from rcspcctivc arcas of the scction was measured with a liquid scintillation counter. The autoradiographic
optical density of thc film was measured with a microdensitometer (Joyce
Locbl 3CS; Gateshead. UK).
Chcmographic artifacts occurring in the autoradiograph were asscsscd
with the sections from an animal untrcatcd with radioactive traccr. Thc
sections were prepared by the same procedures and divided into twogroups.
Onc group was covcrcd with uncxposcd emulsion film to test positive
chcmography, and the other was covered with cmulsion film foggcd by a
10-secstandard exposure to light to test negative chcmography. Both groups
wcrc further subdividcd into three groups and stored separately in lighttight boxes. One of these groups was exposed for 5 wccks at room tcmpcraturc (about 23'C); the others wcrc exposed for 5 and 10 wccks at 4'C andfor 10 wccks at - 2O'C. After mposurc, the films were developed for 5 min
at 20'C. fixcd, and mounted under a coverslip with glycerin. and wcrc cxamincd by light microscopy.

Results
Quality of Emulsion Film
The quality of the prepared emulsion film used in the light microscopic autoradiography is shown in transmission electron micrographs (Figure 2). The photographs show that the thickness of sin(Figure 2A). Figure 2B shows two
gle layer was about 1
thicknesses of the film, and two layers of the film were brought
into full contact. The silver halide grains were distributed uniformly
in the film (Figure 2C). Figure 3 shows the results ofstandard s a m ples exposed for 3 days. The intensity of the autoradiographic optical density and the radioactivity showed an approximately proportional relationship when the proper exposure period was selected(Figure 3).

AUTORADIOGRAPHY OF WATER-SOLUBLE MATERIALS

1807

A

I
0.02

0.04

0.06

0.08

Autoradiographic optical density
Figure 3. Relationshipbetween radioactivity and autoradiographic optical density of emulsion film developed. A single layer (A) (about 1 pm thick) or (E)double layers (about 2 vm thick) of emulsion film were exposed on samples containing various amounts of '%a.

I

Figure 2. Electron micrographs of an emulsion film. A and B show crosssections of a single and double layer of emulsion film prepared from one andtwo bubbles, respectively,and C shows the distribution of silver halide grainsin the emulsion film prepared from one bubble. Bars = 2 pm.

Autoradiography
The following results were accomplished in serial whole-body sections of 6 pm thickness. Figure 4A shows an HE-stained serial section adjacent to the section in Figure 4B. Figure 4B shows the wholebody autoradiograph obtained from a section of a 9-day-old rat
sacrificed 10 sec after an IV injection of 45CaC12. and Figure 4C
shows an autoradiograph which was exposed for a shorter period
than Figure 4B,because the radioactivity in the organs within theabdominal cavity is very intense. Tissues and organs were readily
distinguished and well preserved. The tracer was also well localized. The highest radioactivity was found in the heart and bloodvessels. and the radioactive calcium had already circulated aroundthe body. The calcium was incorporated into the hard tissues suchas bone, dentin, and enamel. High radioactivity was revealed in
the lung, parenchyma of the liver, and in submandibular and labial
glands. In tooth germ, the activity in the stellate reticulum wasmuch higher than that in the dental papilla. The activity in brain

and fat tissue was very low. No autoradiographically detectable radioactivity was found in the urine or in the contents of the small
intestine and stomach. Detailed localization of the radioactivity
was examined in whole-body sections 3 Fm thick, and the distribution in the long bones and the cortex of kidney are shown asa demonstration in Figures 5 and 6. Both autoradiographs showedthat the tracer was well localized in the tissues. In the bone, very
high radioactivity was observed on the surface of trabeculae andextracellular spaces, whereas the radioactivity in osteoblasts adjacent to the trabeculae and other cells was very low (Figures 5A and
5B), and the radioactivity in the cortex of the kidney was observedin the glomerulus and renal tubules (Figures 6A and 6B).

Cbemograpby
Figure 7 shows the positive chemography caused at 23°C or - 20°C.
The chemography appeared in most of the tissues in the sections
kept for 5 weeks at about 23°C. Intense chemography was particularly observed in the retina, bone, cementum, dentin, and enamelof the forming side of the lower and upper incisor at low magnification (Figures 7A and 7B). The chemography was significantly
decreased when the sample was kept for 5 weeks at 4'C, and was
not recognized in these tissues. However, the chemography was observed at high magnification when the sample was kept for 10weeks.
Intensive chemography was seen over the cells of several tissues suchas bone marrow, spleen, lung, liver, muscle, and tooth germs. Although the chemography caused by most of these tissues was notapparent when the sample was kept for 10 weeks at -2O'C. thecells of lung (Figure 7C), bone marrow (Figure 7D), the papillary
layer of tooth germs (Figure 7E), and muscle (Figure 7F) still caused
chemography.
Figure 8 shows the negative chemography caused at 4'C and-2o'c. When the samples were kept for 5 Weeks at 23"C, mosttissues showed negative chemography. Although the chemography
was significantly decreased when the sample was kept at 4'C or- 20"C, it was recognized in some areas. The most intensive negative chemography was observed in the blood, the feces in the large

1808

KAWAMOTO.dP

Figure 4. Serial whole-body sections of a 9-day-old rat sacrificed 10 sec after an IV injection of ''CaC12. (A) Section stained with hematoxylin and eosin. (E,C).
Wholebody autoradiograph of the same section; C was exposed for a shorter period than E.White areas in the autoradiograph correspond to high radioactivity.
SR. stellate reticulum; DP, dental papilla. Bar = 5 mm.

I#

Ah

I

Figure 5. Light microscopic autoradiographs of long bone of the same animal used for the whole-body autoradiography. (A) Trabeculae in calcification zone;(8) the portion indicated with an a r m in A. Arrowheads in A indicate the surface of bone, and arrows in B indicate the cells. Bn, bone; Os, osteoblast; *, cells.
Bars = 10pm.

L
A

L A
b

c
Figure 6. Light microscopic autoradiographsof the kidney of the same animal used for the whole-body autoradiography.(A, E) The glomerulus and proximal renal
tubules of kidney. A is focused on the silver grains and B is focused on the tissue. Rt. renal tubule; GI, glomerulus. Bar = 10 r m .

1810

AUTORADIOGRAPHY OF WATER-SOLUBLE MATERIALS

1811

Figure 7. Positive chemography. (A, E) Samples were kept for 5 weeks at about 230 C; (C, D, E, F) samples were kept for 10 weeks at -2OOC. (A) Eyeball; (E)proximal side of lower incisor; (C) lung; (D) bone marrow; (E) papillary layer in the zone of presecretion and initial secretion of ameloblasts in a lower incisor;(F) cross-section of tendon muscle. A and B are transmission and C, D, E. and F are darkfield illuminations. Arrowheads in D indicate the surface of the bone.
Re. retina; Bn, bone; BM. bone marrow; En, enamel; Dn, dentine; Cm. cementum; Am, ameloblastic layer; PL. papillary layer; Os. osteoblast; *, muscle fiber.
Bars; A = 1 mm; B = 0.2 mm; C-E = 33 pm.

intestine, and the goblet cells of the intestine (Figures 8A and 8B).
In addition, intense chemography was detected over the enamelin the maturation phase and appeared to relate to the cyclical activity of the ameloblastic layer of this phase (Figure 8A). In long

<
.

-

.,.

.

.

bone, relatively intense chcmography was observed in the calcification zone of bone. Chemography was also observed in cells of
the bone marrow (Figure 8C), liver (Figure 8D), proximal tubulesof kidney (Figure 8E), and spleen (Figure 8F).

-

.

' I

,

.:I.

,

.;

. :....
I:

Figure 8. Negative chemography caused on fogged emulsion film. A, B Samples were kept for 10 weeks at 4OC; (CF)samples were kept for 10 weeks at -2OOC.
White areas correspond to intensive negative chemography. (A) Upper incisor; (E) abdominal cavity; (C) bone marrow; (D) liver; (E) cortex of kidney; (F)spleen.
Arrowheads in A indicate the blood vessel, and arrows in A and B indicate cracks in the emulsion film. *, small and large intestine; BM. bone marrow; Bn. bone.
Bars: A,B,D,F = 0.5 mm; C,E = 100 pm.

KAWAMOTO

1812

Discussion
When the distribution of drugs or metabolites in the body is studied by autoradiography, it is often necessary to detect not only the
distribution of water-soluble tracer but also the early changes intheir distribution after administration of the tracer. With respect
to the whole b o d y level, the tape sectioning technique initiatedby Ullberg (23) in 1954 has been widely used as the standard technique (12.25). For light microscopic autoradiography, various attempts have been reported by many researchers (1.6,7,11,17,20,
21.24.26.27).

Kawamoto and Shimizu (9) dexribed procedures for the preparation of frozen sections mounted on a glass slide and for the application of a dried thin film of autoradiographic emulsion on thesection. Their method is available to examine early changes in the
distribution of tracer, but it is not adequate to demonstrate theprecise location of tracer at high magnifications because the sections routinely prepared by the method were too thick (about 10pm) for detailed observation (10). To make thin, well-preserved
whole-body sections, it was necessary to select a sharp knife andsection supporting material. In the present work, a disposable microtome blade of the Feather type was used, and the blade holder forthe cryomicrotome (LKB; 2258 PMV)was specially designed andmade of stainless steel. The blade can be attached to the holder
and changed to a new,blade without any alteration in the sectioning conditions. By use of this type of blade, whole-body sectionsof 2 pm thickness can be cut, although the quality of the section
is influenced by the shape and size of the CMC block. The width-

Figure 9. (A-C) Small intestine; (D.E) calcification zone of long bone. (A) HE staining; (8)Alcian blue staining at pH 25; (C.E) alkaline phosphatase activity; (D)toluidine blue staining. Osteoblasts are located on the bone surface in D and E. Bn. bone. Bars
10 r m .

AUTORADIOGRAPHY OF WATER-SOLUBLE MATERIALSof the block facing the knife edge can significantly affect the quality, whereas the cutting distance has a lesser influence on the qualityof the section. The best sections can be cut from a narrow block,and the quality of the section is much better than that cut witha conventional microtome knife. The plastic block usually used toprevent the separation between section and tape at the knife edge
during cutting is not advisable, because the pressure involved affects the thickness of the resulting section.
As a supporting material, a film that is thin, flexible, and transparent, with high adhesive power, is the most desirable for this purpose. Five kinds of adhesive tape, such as Scotch 810 (4), adhesive
cellophane tape, and cellophane, polyethylene and polyvinylidene
chloride film (8) coated with synthetic adhesive glue, were tested.
Among them, commercially available Scotch 810 and cellophanetape were not suitable because their adhesivity was not great enoughto preserve the detailed structure of the section, and cellophane
film could not be used because the film was influenced by the solvents during the staining process. Although both polyethylene andpolyvinylidene plastic films fit the purpose, polyvinylidene chloride film is much better than polyethylene film because the latteris easily stretched.
The combination of a disposable microtome blade and Saran
Wrap adhesive tape (polyvinylidene chloride film) permits frozen
sections 2-20 pm thick to be cut, and more than 95% of the wholebody serial sections of 5 pm thickness from a block size of 4 x
12 cm were satisfactorily preserved. The sections can be used in almost all histological and histochemical studies because the adhesive tape supporting the section is not affected by the staining
processes. Histological demonstration is shown in Figures 3A and
9A-9D, and the appearance of tissue is superior in comparison withthose previously recorded by the author (9). In addition, the distribution of mucopolysaccharide demonstrated by the method of
Mowry (14) and of alkaline phosphatase activity demonstrated by
the method of Burstone and Keyes (2) is shown in Figures 9B. 9C.
and 9E. That the enzymatic activity is localized on the cell membranes of the osteoblasts is clearly shown. When the technique is1813applied to make whole-body sections of, for example, an adultmouse, one may encounter difficulty in cutting the fully calcified
tissues, such as enamel and dentin. In this case, it is recommended
that such hard tissues be ground away from the cutting surface of
the CMC block using a dental burr. A whole-body section of an
adult mouse is shown in Figure 10. The organs and tissues are wellpreserved. Although ice crystal artifacts can be minimized by accelerating the rate of freezing, this artifact never can be avoided
in such large samples as young rats or adult mice. In fact, the histology, especially in the middle part of the section, revealed damage
to the detailed cell structure, but the morphology was preserved
well enough for cell identification.
For light microscopic autoradiography, soluble tracers can diffiise during the autoradiographic procedure, such as during mounting of the emulsion and exposure. Various emulsion films(1.3,9,13,16.22) and methods (1,5,9,11.13.22,26) for mounting thefilm have been reported, and stripping film and the photographic
emulsion coated on a glass slide have been most widely used. However, as the results of various trials, the film formed by a bubbling
technique was most suitable for covering a large area and for enabling a close contact between the sections without serious diffusion of water-soluble tracer (9). In this study, sections of 3 pm thickness were prepared and emulsion films of 1 pm thickness were
mounted on the section. The light microscopic autoradiograph thus
prepared demonstrated that 45Cawas well localized in the tissues(Figures 5 and 6). It can be readily observed in the autoradiographthat the radioactivity in the osteoblasts and cells in the space between the trabeculae was very low, although in the extracellularspaces and the surface of the trabeculae it was very high.
It has been known that latent images can be created in autoradiography by direct chemical action of the specimen on the emulsion(15,18). These artifactscan be recognized by examining control specimens identical to the experimental ones but non-radioactive, exposed to emulsion in the same way. Roger and John (19) havereported that the most intensive negative chemography occurs overthe salivary gland and pancreas. In the present study, positive andFigure 10. Whole-body section of 12-week old mouse stained with HE. Enamel and dentine was ground off with a dental burr. Bar = 5 mm.

1814

negative chemographies at different temperatures were examinedusing fresh whole-body sections. When the samples were exposedat room temperature, many tissues caused scvcre positive and/or
negative chemography. Although these chemographies were significantly reduced when the sample was exposed at low temperature (4’C or - ZO’C), the chemographies were still detectable overthe cells of several tissues; relatively intense positive chemography
was found in the bone marrow, lung, and muscle (Figures 7C-7F),
and negative chemography was also recognized in blood, spleen,bone marrow, proximal tubule of kidney, and feces of the large intestine (Figure 8). In my experience therefore, when the distribution of tracer in the tissues showing intense chemography is to bestudied, I recommend exposure for a period of less than 5 weeks
at below -20’C.
Acknowledgments
I wsh to express my gratatude to Pmfssor M. Shimizufor frequent stimuhting and helpficl discussions. I am also indebted to Professor R. U? Fearnhad for his valuable comments and advice.

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