Histochem Cell Biol (2000) 113:331–339
Digital Object Identifier (DOI) 10.1007/s004180000149

O R I G I N A L PA P E R

Tadafumi Kawamoto · Masaharu Shimizu

A method for preparing 2- to 50-µm-thick fresh-frozen sectionsof large samples and undecalcified hard tissues

Accepted: 13 March 2000 / Published online: 11 April 2000
© Springer-Verlag 2000

Abstract This article describes a method for preparing
2- to 50-µm-thick fresh-frozen sections from large samples and completely calcified tissue samples. In order toperform the more routine work involved, a tungsten carbide disposable blade was installed to a heavy-duty
sledge cryomicrotome. An entire 10-day-old rat andbone and tooth samples from a 7-month-old rat were rapidly frozen. The frozen samples were attached to the
cryomicrotome stage. The cutting surface of the samples
was covered with a polyvinylidene chloride film coatedwith synthetic rubber cement and cut at –25°C. The soft
tissues and the hard tissues were satisfactorily preserved
and all tissue cells were easily identifiable. Enzymatic
activity in the fresh sections was much stronger than thatin chemically fixed and/or decalcified sections. The sections permitted histological and histochemical studies
without trouble. In addition, the sections can be used for
multiple experiments such as immunohistochemistry, insitu hybridization, and electron microprobe X-ray microanalysis. This method can be used with conventional
cryomicrotome equipment.
Key words Fresh-frozen section · Whole-body section ·Hard-tissue section · Enzyme histochemistry · Bone ·ToothIntroduction
The relationship between tissue structure and function
has been studied using many different methods. Thesemethods include histology, general histochemistry, enzyme histochemistry, immunohistochemistry, in situ hybridization, autoradiography, and electron microprobe Xray microanalysis. When such studies are carried out
T. Kawamoto (✉) · M. Shimizu
Department of Biochemistry, School of Dental Medicine,
Tsurumi University, 2–1-3 Tsurumi, Tsurumi-ku, Yokohama,Japane-mail: k01557@simail.ne.jp
Tel.: +81-45-5811001 Ext. 3448, Fax: +81-45-5739599

with serial sections, the results are easily and accurately
correlated with each other and function can be describedin great detail.
The tissues are usually chemically fixed to preserve
the cell structure with minimum alteration from the living
stage. In calcified tissues, decalcification is performed topermit easy sectioning. Fixation and decalcification usually result in a reduction of enzyme activity and immunoreactivity (Baker et al. 1958; Takeshita et al. 1983;
Mullink et al. 1985; Mukai et al. 1986; Van Noorden andVogels 1986; Wakisaka 1986; Van Noorden et al. 1989;
Van Den Munckhof et al. 1994; Fukase 1997). In addition, there is an accompanying loss and/or dislocation ofwater-soluble materials from the tissue.
These problems are prevented by preparing thin sections without fixation and decalcification. Many attempts
to prepare such sections have been reported. Methods
used are summarized as follows:
1. Coating the frozen cutting surface with a polymeric
solution before each sectioning process (Fitz-William
et al. 1960; Fink 1986, 1992; Aaron and Carter 1987;
McElroy et al. 1993)
2. Covering the frozen cutting surface with a sheet ofpaper (Watanabe et al. 1978; Kihara 1984; Kawamotoand Shimizu 1986; Hill and Elde 1990; Shimada and
Watanabe 1995)
3. Covering the frozen cutting surface with pressuresensitive adhesive tape (Parmgren 1954; Ullberg
1954; Farebrother and Woods 1973; Fukuda andShindo 1974; Watanabe et al. 1975; Deak et al. 1976;
Rijntjes et al. 1979; Larsson and Ullberg 1981; Sjogren et al. 1981; Hammerstrom 1986; Kawamoto andShimizu 1994).
Only the third method developed for whole-body autoradiography (Ullberg 1954) satisfies our requirements.
However, the quality of the section is not suitable for microscopic examination. Kawamoto (1990) modified thetechnique and showed that the sections are very usefulfor histological and histochemical studies at the microscopic level. However, preparing thin sections, such as a

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2- to 5-µm-thick section, of completely calcified bone
and tooth sample had not yet been achieved. We are
studying the calcification of hard tissue, being especiallyinterested in calcium transport to the mineralizing hardtissues. Therefore, we need sections thin enough to be
used either for studies on water-soluble materials or for
enzyme histochemistry at the microscopic level.
This paper describes a method for preparing 2- to
50-µm-thick fresh-frozen sections either from a large
sample or from completely calcified tissues. Histologicaland histochemical demonstrations are included.

Materials and methods
Preparation of frozen sections
We modified some sectioning conditions and procedures in themethod described by Kawamoto (1990) and used improved equipment. A 10-day-old rat (weighing approximately 22 g) was usedas the large sample. A 7-month-old rat (weighing approximately
350 g) was used as the completely calcified tissue sample. The entire 10-day-old rat was frozen in hexane (–94°C) cooled with acooling apparatus (Neocool Bath; Yamato, Japan). The adult rat
was anesthetized with sodium pentobarbital. One milliliter 0.04%
calcein (Dojindo Laboratory, Japan) in physiological saline wasinjected into the abdominal cavity. After 2 h, the thighbone andthe lower jaw were dissected and frozen in liquid nitrogen. The
frozen sample was immersed in a stainless steel container filledwith a 5% carboxymethyl cellulose (CMC) gel [the container was
selected to fit the size of the sample (Fig. 1A)]. The container wasplaced in the cooled hexane and the CMC gel was completely frozen (the upper side of the CMC gel in the container must not be
immersed in the coolant to avoid cracks during freezing). The frozen CMC block was attached to the sample stage of the cryomicrotome (CM 3500; Leica Instruments, Germany) in the cryochamber (–25°C; Fig. 1B).
After sitting for 2 h, the block was trimmed with a disposable
tungsten carbide blade (Jung TC-65, 35° angle; Leica Instruments)
having a clearance angle of 5°. The surface was covered with a
polyvinylidene chloride film (10 µm thick; Asahikasei Kogyo, Japan) coated with synthetic rubber cement. The sample was cut at a
speed not exceeding 4 mm/s. The sectioned side of the film section was placed on the flat metal plate cooled in the cryochamber
and pressed with a rubber roller to ensure close contact. The film
section was then fixed on a cooled glass slide with double-sided
adhesive tape.
We recommend the 5- to 10-µm-thick sections for macroscopic
examination and the 2- to 5-µm-thick sections for microscopic examination. The former application requires the following procedures:
1. Freeze-drying the frozen sections in the cryochamber (–25°C)
for approximately 12 h
2. Placing the dried sections in a box containing silica gel toavoid condensation
3. Removing the sections from the cryochamber
4. Immersing the sections in 100% ethanol to remove trapped air
bubbles.
The latter application requires the following procedures:
1. Removing the frozen sections from the cryochamber
2. Momentarily thawing of the sections
3. Immersing the sections in 100% ethanol at room temperature.
Histological examination
For histological staining, the sections in 100% ethanol wereplaced in a 3% glutaraldehyde solution buffered with 0.1 M phos-

Fig. 1 A Stainless containers for embedding the frozen sample.
B Cut surface of the sample, frozen section (arrow), and knife
stage

phate (pH 7.2) and fixed for 5 min. The sections were then stainedwith 0.1% toluidine blue for 10–30 s, rinsed with running water
for about 5 min, and mounted with glycerin under a cover glass.
For alizarin red S staining, freeze-dried sections immersed in
100% ethanol were used. The sections were stained with 1% alizarin red S for about 3 min and mounted with glycerin under acover glass.
When the large section is mounted, the polyvinylidene chloride
film supporting the section is used as a cover. The sectioned sideof the film section is turned toward the glass slide coated with30% glycerin and thus attached to it. Excessive glycerin is removed with a sheet of filter paper and the glass slide is left for
about 2 days. The section is sandwiched between the supporting
film and the glass slide and is permanently preserved.
Calcein fluorescence examination
The thighbone freeze-dried section was placed in a box containing
silica gel and removed from the cryochamber. The section wasplaced on a glass slide and protected with a cover glass. There wasno mounting medium. Calcein fluorescence was observed with afluorescence microscope (Olympus, Japan). A blue beam, between
450 and 495 nm in wavelength, was used for calcein excitation.
An emission filter, which permits the passage of wavelengths exceeding 510 nm, was used for observation.

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Fig. 2A–D A 2-µm-thick section of the 10-day-old rat (toluidine
blue staining). A Whole body. B Molar. C Cerebellum. D Small
intestine
Effects of fixation and decalcification
To study the effects of fixation and decalcification on enzyme activity, alkaline phosphatase (ALPase) was examined. The fresh
sections were used as a control. To examine the fixation effect, thefresh sections were fixed with a 4% paraformaldehyde (PFA)
0.1 M phosphate buffer (pH 7.4) at room temperature for 2, 6, 12,
and 24 h. To examine the decalcification effect, the fresh sections
were decalcified with 5% EDTA adjusted to pH 7.0 for 6 days. For
both examinations, the fresh sections were fixed with PFA for 2 h
and decalcified with EDTA for 6 days. Two sections were used ineach examination. The ALPase activity was demonstrated using
the method described by Burstone (1958). Each section was incubated at room temperature for 2 min and either mounted withglycerin under a cover glass or sandwiched between the film andthe glass slide.
Photographs
Photographs were taken with a digital camera (Fujix HC-300; Fuji
Photo Film, Japan) connected to a desk-top computer. The resulting images were reproduced using PhotoShop (Adobe Systems,
USA) and printed with a color printer (Pictrography 4000; Fuji
Photo Film).

Results
The entire 10-day-old rat and the bone and tooth samplesof the 7-month-old rat were easily cut into 2- to 50-µm-

thick sections. All sections shown in this paper are 2 µm
thick. Figure 2A shows the whole-body section stainedwith toluidine blue. Figure 2B–D shows the upper molar,
cerebellum, and intestine at high magnification. Soft tissues such as the brain, muscles, glands, intestine, lung,
liver, pancreas, kidney, and spleen were perfectly preserved. Blood remained in the heart and blood vessels. In
addition, the calcifying bone, dentine, and enamel were
almost perfectly preserved. All tissue cells were easily
identified at high magnification (ameloblast and odontoblast in the tooth germ, epithelial cells in the small intestine, mesangial cells in the kidney, and glandular cells in
the glands).
Sections prepared from the adult rat thighbone areshown in Figs. 3 and 4. Portions of the skeletal muscle
were damaged during dissection, but the remaining
skeletal muscle, ligament, and bone marrow were satisfactorily preserved (Fig. 3A). In the diaphysis, the outerand inner basic lamellae were easily distinguished. The
alizarin red S-stained section showed that the calcified
bone was almost completely preserved (Fig. 3B). Figure 4C shows the epiphysis portion of Fig. 3A (arrow)at high magnification. Some small cracks caused by
cutting were observed in the bone at high magnification, but topographical and structural relationships between the calcified tissues and the soft tissues were wellretained. The osteoblasts were clearly seen on the bone
surface. In the bone marrow, many types of cells suchas myeloblasts and eosinophilic leukocytes were identified.

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Fig. 3A,B A 2-µm-thick serial sections of the 7-month-old rat
thighbone. Insets of the micrograph show magnified views of theareas indicated with each arrow. A Toluidine blue staining. B Alizarin red S staining. A Area of epiphysis shown at high magnification in Fig. 4C

Calcein fluorescence in the thighbone serial sections
is shown in Fig. 4A,B,D. Fluorescence appeared on mostof the bone surface in the freeze-dried sections (Fig. 4A).
Double fluorescent lines were noticed on some parts ofthe surface layer (Fig. 4D). Very intense fluorescence
was observed in spots on the bone surface layer. In the

fixed stained section, fluorescence was significantly reduced (Fig. 4B).
Figure 5 shows the lower jaw serial sections of theadult rat. The soft tissues and the hard tissues were satisfactorily preserved (Fig. 5A,B). In the molar, the dentine
and the cementum were perfectly preserved and the noncalcified areas in the dentine and cementum were clearlyshown (Fig. 5C). The topographical relationship between
the periodontal fibers and the fibroblasts in the periodontium was maintained. In the lower incisor, the enamel
and the dentine were almost perfectly preserved. The
ameloblasts and odontoblasts remained attached to theenamel and dentine surfaces (Fig. 5D).

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Fig. 4A–E Calcein fluorescence in the epiphysis of the2-µm-thick sections preparedfrom the 7-month-old rat thighbone.A Fluorescence in thefreeze-dried section. B,C Fluorescence and light micrographin the toluidine blue-stained
section. D,E Highly magnified
views of the portions indicatedwith the arrows in A and C, respectively. Arrows in D show
the double fluorescence lines.
Bn Bone336

Fig. 5A–D A 2-µm-thick serial sections of the 7-month-old rat
lower jaw. A Toluidine blue staining. B Alizarin red S staining.
C,D Magnified views of the portion indicated respectively by thearrows A and B in A. Insets of C and D show periodontium andameloblast layer. Arrows in B and C indicate the root of the molar
and fibroblasts, respectively. Bn Bone, Dn dentine, PDn predentine, Cm cementum, PO periodontium, En enamel, Am ameloblast,
PL papillary layer

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Fig. 6A,B Alkaline phosphatase activity (shown in blue) in the
10-day-old rat molar tooth. A Fresh section. B Section fixed with
4% paraformaldehyde for 2 h and decalcified with 5% EDTA for
6 days. Arrows in B indicate the small intestine

ALPase activity in the fresh section was very strong
and the reaction products shown in Fig. 6A were produced within 2 min. We confirmed the observation ofmany other researchers that ALPase activity is distributed through many tissues (kidney, intestine, bone, toothgerm, and glands) and that it is localized on the cell
membrane. The 4% PFA fixation and the 5% EDTA decalcification reduced ALPase activity. ALPase activity
on the kidney, the bone, the tooth germ, and the gland
was reduced conspicuously, as shown in Fig. 6B, when
the sections were fixed with the PFA for 2 h and decalcified with EDTA for 6 days. But the activity on the small
intestine remained.

Discussion
The 2- to 50-µm-thick sections were prepared routinely
from large samples and from completely calcified bone
and tooth samples. In order to achieve this, some refine-ments were made in the equipment. A disposable tungsten carbide blade was used and steps were taken to
avoid temperature increases in the chamber. A superhard reusable knife made of a hardened tungsten steel
permits cutting of hard tissues such as bone, dentine,and enamel (Van Noorden and Vogels 1986; Hill andElde 1990; Haines 1992; McElroy et al. 1993; Nakamura
et al. 1994). However, even a knife made of such a hardmaterial loses sharpness when cutting completely calcified hard tissues. A disposable blade made from special
high-grade tungsten carbide is also able to cut hard tissue. A sharp knife is indispensable to a high quality section, so we designed a holder to use a disposable knife.
The blade can be moved from side to side in the holder
so that the samples are always cut with a sharp knifeedge. Another advantage of the disposable blade is theability to change blades without changing the conditionsof an experiment. A disposable blade is also economical.
Doing handwork in a cryochamber with a large lid
causes an undesirable temperature increase, particularlyin the upper half of the cryochamber where the handwork is done. To prevent temperature increase, we installed a new lid with a small access window to the cryochamber. A small electric fan mounted inside the cryo-338chamber was used to gently circulate cool air in thecryochamber. These steps minimized temperature increase.
Sections prepared at lower temperatures show excellent histology. However, we were unable to make sections using commercially available adhesive tape such as
Scotch type 688, 800, or 810 at such low temperatures
because the adhesion of the tape deteriorated rapidly
with a drop in temperature. The polyvinylidene chloride
film coated with the synthetic rubber cement maintains
its adhesion at low temperatures and holds and supports
the sections tightly during cutting
Using these improvements, large samples and completely calcified bone, dentin, and enamel samples were
cut without special technical skills and the tissues weresatisfactorily preserved as shown in Figs. 2, 3, 4, and 5.
Almost all 3-µm-thick sections were satisfactorily preserved. These sections withstood immunohistochemicaltreatment for 4 days. There was no deterioration even after leaving the sections in 100% ethanol for more than
1 month.
Ice crystal formation damages tissue and appears conspicuously in the central portion of the large samples.
This damage can be minimized by using hexane containing solid hexane (–94°C) or liquid nitrogen instead ofdry-ice hexane. This is especially true when dissected
and trimmed samples are used. Damage can be further
minimized by using liquid propane. However, there appears to be no way to avoid it completely. We found thatthe ice crystals could be eliminated by thawing the frozen section immediately after cutting and that this technique considerably improves histological views. We recommend this technique when the section is examined athigh magnification.
Either fixation or dehydration as shown in Fig. 4 results in fluorescent dye loss in the bone. It also decreases
enzyme activity (Van Noorden and Vogels 1986; Wakisaka 1986; Van Den Munckhof et al. 1994). Our technique (shown in Figs. 4, 6) eliminates both these problems. Though the dates are not shown, the immunoreactivity of the fresh section is better than that of fixed
and/or decalcified sections.
This technique can be used with conventional cryomicrotome equipment. Sections can be used for many typesof research including characterizing enzyme, antigen,mRNA, and water-soluble molecule (or element) distribution during growth and development of general bodytissue. When such research is done using serial sections,the technique is a very powerful tool.
Acknowledgement We are grateful to Dr. Fabio Henrique de S.L.
Pinheiro (San Paulo University, Brazil) for his generous assistance.

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