Volume 45(2): 307–313, 1997

The Journal of Histochemistry & Cytochemistry

TECHNICAL NOTE

Embedding of Bone Samples in Methylmethacrylate:

An Improved Method Suitable for Bone Histomorphometry,
Histochemistry, and Immunohistochemistry

Reinhold G. Erben
Institute of Physiology, Physiological Chemistry, and Animal Nutrition, University of Munich, Munich, Germany

SUMMARY Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a

technique widely used for bone histomorphometry. However, conventional MMA embed-
ding causes almost complete loss of enzyme activity and protein antigenicity in the tissues.
Recently, an MMA embedding technique has been reported that preserves enzyme activity
and antigenic determinants in bone tissue. We describe here a modification of this embed-
ding method. For our modified MMA embedding process, commercially available meth-
acrylates can be used without purification, and the histologic quality of bone sections is
comparable to that of conventionally MMA-embedded bone specimens. The technique re-
ported here can be employed for embedding of larger bone samples and is suitable for his- KEY WORDS
tochemical and immunohistological applications as well as for routine bone histomor- Methylmethacrylate
phometry. By addition of methylbenzoate during infiltration and polymerization of the Methacrylates
plastic, the antigenicity of the tissue was improved. As applications of this novel technique, Bone
demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as Rats
positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sec- Histomorphometry
tions of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method de- Histochemistry
scribed here might be useful for the inclusion of histochemical and immunohistological Immunohistochemistry
methods into bone histomorphometry. (J Histochem Cytochem 45:307–313, 1997) Methylbenzoate

Methylmethacrylate (MMA) embedding of unde-
calcified bone biopsies is a technique widely used for
bone histomorphometry and bone marrow hematopa-
thology, both in clinical medicine and in animal exper-
imental studies (Frisch et al. 1985; Schenk et al. 1984;
Baron et al. 1983). Compared to other plastic embed-
ding media such as water-soluble methacrylates (e.g.,
glycolmethacrylate, GMA), MMA offers a variety of
advantages. (a) MMA penetrates tissue better than
GMA. This is of importance especially for larger bone
specimens such as rat tibiae or vertebrae or human
transiliac biopsies, and the histological quality of bone
sections is generally higher in MMA-embedded bone
samples compared to GMA-embedded specimens. (b)
Correspondence to: Reinhold G. Erben, Inst. of Physiology,
Physiological Chemistry, and Animal Nutrition, U. of Munich, Vet-
erinärstr. 13, 80539 Munich, Germany.
Received for publication June 14, 1996; accepted September 18,
1996 (6T4007).
GMA is a bifunctional molecule and forms crosslinks
during polymerization of the plastic. Therefore, re-
moval of the resin from tissue sections is not possible
for GMA-embedded material. MMA, on the other
hand, can easily and completely be removed from tis-
sue sections. This results in superior staining charac-
teristics and excellent morphological detail. However,
conventional MMA embedding causes almost com-
plete loss of enzyme activity and protein antigenicity
in the tissues, and therefore precludes the use of his-
tochemical and immunohistological methods.
Polymerization of methacrylates is an exothermic
radical chain reaction. Conventional MMA mixtures
mostly use organic peroxides as initiators of polymer-
ization, and polymerization is usually carried out at
temperatures between 30 and 45C, using thermal de-
composition of the peroxide to start the polymeriza-
tion process (Schenk et al. 1984; Baron et al. 1983). It
is believed that enzyme activity and antigenic determi-

© The Histochemical Society, Inc. 0022-1554/97/$3.30 307

308
nants are lost because of temperature peaks and modi-
fication of chemical structures in the tissue by reactive
radicals formed during polymerization of MMA (Baskin
et al. 1992; Wolf et al. 1992). It is also possible to ini-
tiate polymerization of methacrylates by blue or uv
light photons. This technique allows polymerization at
low temperatures (14 to 230C), and it has been
shown that enzyme activity and antigenic determinants
are well preserved during low temperature, light-initi-
ated methacrylate embedding (Baskin et al. 1992;
Tenorio et al. 1992).
In vivo labeling of bone with fluorochromes (e.g.,
tetracyclines) is a widely used method to assess min-
eral apposition rate and bone formation rate in bone
histomorphometry. Fluorochromes are calcium-seek-
ing molecules that bind to the mineralization fronts in
bone formation sites. In sections of undecalcified bone,
fluorochromes can be visualized by their specific fluo-
rescence under uv or blue light excitation. Prolonged
exposure to blue or uv light results in irreversible fad-
ing of fluorochromes bound to bone mineralization
sites. Therefore, possible fading of fluorochromes pre-
cludes the use of light for polymerization of the meth-
acrylate in a routine plastic embedding of undecalci-
fied bones suitable for both bone histomorphometry
and histochemical and immunohistological techniques.
Recently, an MMA embedding technique has been
reported that can be used for embedding of larger
bone samples, and which preserves enzyme activities
and antigenic determinants in bone tissue (Wolf et al.
1992). This embedding mixture polymerizes at 215 to
220C. It employs benzoyl peroxide as initiator and
the aromatic amine N,N-dimethyl-p-toluidine as ac-
celerator for chemical polymerization at low tempera-
tures. However, the described MMA embedding tech-
nique involves tedious purification and destabilization
of the methacrylate components, and the histological
quality of the sections, in our hands, is inferior in
comparison to specimens embedded with conventional
MMA techniques.
We report here a modification of the embedding
technique originally described by Wolf et al. (1992).
For this modified embedding process, commercially
available methacrylates can be used without purifica-
tion, and the histological quality of bone sections is
comparable to that of conventionally MMA-embedded
bone specimens. As examples of two histochemical re-
actions relevant to bone research, positive staining of
osteoblasts for alkaline phosphatase (AP) and of os-
teoclasts for tartrate-resistant acid phosphatase (TRAP)
in bone sections is shown. As example of an immuno-
histochemical reaction, positive labeling of rat mac-
rophages and osteoclasts with the monoclonal anti-
body (MAb) ED1 is demonstrated in sections of rat
liver and bone.
Erben
Materials and Methods
Animal Procedures
Six 3-month-old female Fischer-344 rats (Charles River;
Sulzfeld, Germany) were used for this study. The rats were
housed in pairs at 24C and a 12-hr/12-hr light/dark cycle with
free access to tapwater and a commercial rat diet (Altromin
1320; Altromin, Lage, Germany). On Days 14, 9, and 4 before
sacrifice, each rat was injected ip with the fluorochromes de-
meclocycline (20 mg/kg), alizarin complexone (30 mg/kg),
and calcein (20 mg/kg), respectively. All fluorochromes were
purchased from Sigma (Deisenhofen, Germany). After this
labeling protocol, the rats were sacrificed by an ether overdose,
and parts of the liver, the proximal tibiae, and the lumbar
vertebrae were removed. The bones were carefully defleshed
and the marrow cavity in the tibiae was opened by removing
a small part of the lateral condyle, and in the vertebrae by
cutting off the intervertebral discs. The experimental proto-
col was approved by the local government authorities.
Histology
If not otherwise specified, all chemicals were purchased from
Merck (Darmstadt, Germany). All steps of fixation, dehy-
dration, and infiltration were carried out at 4C on a mag-
netic stirrer. The tissue samples were fixed in 40% ethanol
for 48 hrs or in 4% paraformaldehyde (PFA) in 0.1 M phos-
phate buffer, at pH 7.4, for 24 hr. The tissue specimens fixed
in PFA were washed overnight in 0.1 M phosphate buffer,
pH 7.4, containing 10% sucrose. Subsequently, the bone
and liver samples were dehydrated according to the follow-
ing schedule (Schenk et al. 1984): 70% ethanol (2 days),
95% ethanol (2 days), 100% 2-propanol (twice for 1 day),
and xylene (twice for 2 days). After dehydration, the samples
were infiltrated with the plastic embedding mixture using a
three-step protocol. In each MMA solution, the samples
were infiltrated for 3 days. MMA Solution I consisted of 60
ml MMA (Merck; containing 100 ppm hydrochinon) 1 35
ml butylmethacrylate (Sigma; containing 10 ppm hydrochi-
non) 1 5 ml methylbenzoate 1 1.2 ml polyethylene glycol
400. MMA Solution II was a mixture of 100 ml MMA I
with 0.4 g dry benzoyl peroxide, and MMA Solution III con-
sisted of 100 ml MMA I 1 0.8 g dry benzoyl peroxide. All
MMA solutions were stirred for at least 1 hr before use. Po-
lymerization was carried out in 25-ml glass vials (if the sam-
ple size permits, smaller glass vials can be used without mod-
ification of the method). The infiltrated tissue was placed on
a polymerized layer of plastic in the bottom of the glass vials.
To prepare the plastic bases, 600 ml of N,N-dimethyl-p-tolui-
dine was added to 100 ml of cold (4C) MMA III and stirred
for a few minutes. Immediately thereafter, 5 ml of the poly-
merization mixture was poured into each glass vial. Each
vial was then thoroughly gased with N2 or CO2, capped, and
polymerized within 1 day at 4C. The polymerization process
was sensitive to oxygen, and bases did not polymerize in the
presence of oxygen. The glass vials with the plastic bases can
be stored for years. To prepare the polymerization mixture,
400 ml of N,N-dimethyl-p-toluidine was added to 100 ml of
cold (4C) MMA III, and stirred for a few minutes. After ad-
dition of the accelerator, care was taken that the polymeriza-
tion mixture was kept cold at all times. After the infiltrated
Methylmethacrylate Embedding Technique
tissue was placed on the plastic layer in the vials, the vials
were completely filled with polymerization mixture (about
20 ml) to exclude air, capped, and transferred to a deep-
freezer. Polymerization was carried out at 218 to 220C and
was complete within 3 days. Polymerized blocks were stored
at 220C.
After trimming of the plastic blocks, 3–5-mm thick sec-
tions were prepared at room temperature with a Microm
HM 360 microtome (Microm; Walldorf, Germany) equipped
with a D profile knife with a tungsten carbide cutting edge.
During sectioning, the knife and the blocks were kept moist
with a sectioning fluid (WIV; Schwetzingen, Germany) or
with 30–40% ethanol. The sections were transferred onto
chromalum–gelatin-coated slides (for conventional staining
and fluorochromes) or to slides pre-treated with 3-amino-
propyltriethoxy-silane (APES, Sigma; for histochemistry and
immunohistochemistry) and carefully stretched using 70%
ethanol. Thereafter, the sections were covered with a poly-
ethylene foil, flattened with a rubber roller, pressed with a
slide press, and dried for 2 days at 42C in the slide press.
For deplasticization, the sections were placed in three
changes of 2-methoxyethylacetate for 20 min each, two
changes of acetone for 5 min each, and two changes of deion-
ized water for 5 min each. Sections were routinely stained
with toluidine blue at acid pH (Baron et al. 1983) and with
von Kossa (Schenk et al. 1984). Sections for analysis of fluo-
rochrome labeling were not deplasticized, were left unstained,
and were mounted with Fluoromount (Serva; Heidelberg,
Germany).
Histochemistry
For demonstration of AP activity, deplasticized sections were
transferred into 0.1 M Tris-HCl buffer, pH 8.2, for 5 min,
and the histochemical reaction was then carried out in 0.1 M
Tris-HCl buffer, pH 8.2, using a commercially available kit
(Vector Red; Vector Laboratories, Burlingame, CA) accord-
ing to the manufacturer’s instructions. Positive staining de-
veloped within 30 min to 2 hr at 37C. Control sections were
incubated in incubation medium without the enzyme sub-
strate. No staining developed in these control sections.
For TRAP histochemistry, deplasticized sections were placed
in 0.1 M acetate buffer, at pH 5.0, for 5 min, and the TRAP
reaction was subsequently performed as described previ-
ously by Baron et al. (1983) using hexazotized pararosa-
niline (Merck) as azo dye and naphthol AS-TR phosphate
(Sigma) as enzyme substrate. Tartaric acid was added to the
incubation medium at a concentration of 100 mM. Positive
staining developed within 15 min to 2 hr at 37C. Control
sections were incubated in incubation solution that did not
contain the enzyme substrate. No staining developed in these
control sections.
Counterstaining for sections stained for AP and TRAP
was performed using Mayer’s hematoxylin (1 min), and the
sections were subsequently mounted with an aqueous mount-
ing medium (Kaiser’s glycerol gelatin; Merck).
Immunohistochemistry
Immunohistochemistry was performed using the alkaline
phosphatase–anti-alkaline phosphatase (APAAP) method.
309
Deplasticized sections were placed in 0.1 M Tris-HCl buffer,
at pH 8.2, for 5 min. After blocking the sections with 10%
rabbit serum (Vector) for 20 min, the sections were incu-
bated overnight at 4C with the MAb ED1 (Serotec; Oxford,
UK) at a 1:100 dilution in Tris-buffered saline (TBS; 0.1 M
Tris, 0.15 M NaCl, pH 8.2) with 1% rabbit serum. Control
sections were incubated with a non-immune myeloma IgG1
(clone MOPC-21; Sigma) at the same concentration as the
ED1 antibody. After washing in TBS containing 0.1%
Tween 20 (three times for 5 min), the sections were incu-
bated for 30 min with rabbit anti-mouse antibody (STAR
37, not crossreacting with rat; Serotec) at 1:25 dilution in
TBS with 1% rabbit serum. After washing (three times for 5
min in TBS with 0.1% Tween 20), sections were incubated
for 30 min with mouse monoclonal APAAP complex (Dako;
Hamburg, Germany) at 1:50 dilution in TBS. Thereafter, the
sections were rinsed (three times for 5 min in TBS with 0.1%
Tween 20), and stained with the Vector Red kit according to
the supplier’s instructions. Levamisole (Vector) was added to
the incubation medium at the concentration recommended
by the supplier. Finally, the sections were counterstained
with Mayer’s hematoxylin for 1 min and mounted with an
aqueous mounting medium (Kaiser’s glycerol gelatin).
Results
The embedding protocol employed in this study re-
sulted in reliable and homogeneous polymerization of
the blocks. The tendency to form bubbles during poly-
merization at the interface between the glass vial and
the tissue surface was reduced when glass vials with a
pre-polymerized base were used. Hardness of the blocks,
sectioning properties, stretching of the sections, and
section quality were comparable to those of conven-
tionally MMA-embedded bone specimens. The hard-
ness of the blocks did not increase even after pro-
longed storage at room temperature (months to years).
The staining characteristics of the sections were excel-
lent. The staining protocols used for sections of con-
ventionally MMA-embedded bones could be used
without any modification. Compared to fixation with
40% ethanol, PFA fixation resulted in better morpho-
logical detail, with little shrinkage of bone marrow
cells (Figure 1). In vivo fluorochrome labeling of the
bones could be visualized by fluorescence microscopy.
The intensity of the fluorescence signals was indepen-
dent of the fixation used and was comparable to that of
specimens embedded in conventional MMA (Figure 2).
Osteoclasts on all periosteal and endosteal bone
surfaces stained positive for TRAP activity (Figure 3).
TRAP-positive cells not in direct contact or in close
proximity to bone surfaces occurred only very rarely
in bone marrow. The intensity of TRAP activity was
about the same in 40% ethanol- and PFA-fixed speci-
mens. AP activity, on the other hand, was more sensi-
tive to different fixation processes and was greatly re-
duced by PFA fixation. Osteoblasts as well as many
310 Erben

Figure 1 Remodeling sites in vertebral trabecular bone of a 3-month-old female Fischer rat. Bone resorption site with osteoclasts above
(arrows), and bone formation site with active osteoblasts on an osteoid seam below (arrowheads). Note good morphological detail, with
minimal shrinkage of bone marrow cells. Five-mm-thick undecalcified section of a PFA-fixed and MMA-embedded first lumbar vertebra.
Toluidine blue stain. Original magnification 3 330. Bar 5 50 mm.
Methylmethacrylate Embedding Technique 311

leukocytes in bone marrow stained positive for AP in
specimens fixed with 40% ethanol at 4C (Figure 4).
Cells positive for AP showed a membranous staining
pattern in most cases. For both TRAP and AP, stain-
ing was absent in control sections incubated in me-
dium lacking the enzyme substrate.
When sections of rat liver were exposed to MAb
ED1, Kupffer cells in liver sinuses were intensely la-
beled (Figure 5A). The liver sections were used as pos-
itive controls for ED1 labeling. In bone tissue, osteo-
clasts on periosteal (Figure 5B) and endosteal bone
surfaces, as well as osteoclasts (chondroclasts) located
beneath the growth plate (Figure 5C), stained strongly
positive with MAb ED1. There were also many ED1-
positive cells in bone marrow. All cells reactive to ED1
showed a typical cytoplasmic staining pattern (Figures
5A–5C). Reactivity to MAb ED1 in liver and bone tis-
sue was observed with about the same intensity for
both ethanol- and PFA-fixed specimens. An isotype-
specific non-immune control MAb used at the same
concentration as the ED1 antibody did not result in
positive staining. At the dilution of primary antibody
used in this study, nonspecific background staining
was almost absent in sections of liver and bone tissue.
Addition of levamisole at the concentration recom-
mended by the manufacturer completely inhibited en-
dogenous AP activity in liver, bone marrow leukocytes,
and osteoblasts in most specimens. In ethanol-fixed
tissue it was sometimes necessary to double the con-
centration of levamisole in the incubation medium to
inhibit endogenous AP activity.
Discussion
Based on the MMA embedding technique described
by Wolf et al. (1992), we have developed an improved
method of MMA embedding suitable for histochemi-
cal and immunohistochemical investigations in bone
tissue as well as for quantitative bone histomorphom-
etry. Compared to the original method, our method is
different in several aspects: (a) Instead of dehydration
of the bone samples with acetone as described by Wolf
et al. (1992), we used a standard dehydration protocol
that is also employed by conventional MMA embed-
ding (Schenk et al. 1984). Although acetone can also
be used for fixation and dehydration of bone samples,
especially for antigens sensitive to formalin or alcohol,
we do not recommend its general use because, in our
hands, acetone fixation causes massive shrinkage arti-
facts and acetone dehydration results in inferior qual-
ity of the bone section. Using the fixation and dehydra-
tion protocol described in this study, our experience,
in general, is that antibodies reactive in paraffin-embedded
material were also reactive in tissue processed with the
MMA embedding technique reported here. (b) The in-
filtration protocol has been changed to achieve opti-
mal infiltration of the tissue with the plastic. Good in-
filtration is the basis for uniform polymerization and
high section quality. (c) The addition of methylben-
zoate to the methacrylate solution during infiltration
and polymerization improved preservation of antige-
nicity of the tissue in our hands. A similar effect has
been described for GMA embedding (Britten et al.
1993), and methylbenzoate also increases survival of
antigens in paraffin-embedded tissue samples after ac-
etone fixation (Sato et al. 1986). In a similar fashion,
the addition of dithiothreitol to all steps of the dehy-
dration, infiltration, and embedding procedure has
been reported to considerably improve survival of an-
tigenic determinants in plant tissue embedded in MMA
(Baskin et al. 1992). Although the exact nature of the
protective effect of these substances during dehydra-
tion, infiltration, and polymerization of the plastic is
not known, this area certainly deserves further study,

Figure 2 Fluorescent micrograph of vertebral trabecular bone of a 3-month-old rat labeled in vivo with the fluorochromes demeclocy-
cline, alizarin complexone, and calcein. The marker interval between the individual labels was 5 days each. Fluorochrome labeling demon-
strates active bone formation in this site (short arrow, demeclocycline; long arrow, alizarin; curved arrow, calcein). Bone appears black (as-
terisk), and cells in the surrounding bone marrow show some autofluorescence. Five-mm-thick undecalcified section, UV/blue-violet excitation.
Original magnification 3 330. Bar 5 50 mm.

Figure 3 Trabecular bone resorption site with TRAP-positive mononuclear and multinuclear osteoclasts (arrowheads) in a PFA-fixed rat
vertebra stained for TRAP and counterstained with Mayer’s hematoxylin. Bone marrow cells not in direct contact with the bone surface do
not show TRAP activity. Three-mm-thick undecalcified section. Original magnification 3 330. Bar 5 50 mm.

Figure 4 Proximal tibia of a 3-month-old rat fixed in cold 40% ethanol. The section is stained for AP and counterstained with Mayer’s he-
matoxylin. Osteoblasts (arrowheads) in the endocortical bone formation site shown and some bone marrow cells are positive for AP. AP ac-
tivity is most intense on the apical cell membrane of the osteoblasts. Mineralized bone appears blue-violet; osteoid is marked with an ar-
row. Three-mm-thick undecalcified section. Original magnification 3 330. Bar 5 50 mm.

Figure 5 Liver (ethanol-fixed) and tibiae (PFA-fixed) from 3-month-old rats labeled with MAb ED1 (APAAP method). Labeling with ED1 is
intense on Kupffer cells in liver sinuses (A), on osteoclasts (arrows) in a periosteal bone resorption site (B), and on osteoclasts (chondroclasts,
arrowheads) located beneath the growth plate (C). Sections are counterstained with Mayer’s hematoxylin. Addition of levamisole to the in-
cubation medium completely inhibits AP activity in liver cells and osteoblasts (arrows) in the primary spongiosa of the proximal tibial meta-
physis, and almost completely in growth plate (asterisk) chondrocytes. Mineralized bone and calcified cartilage appear blue-violet. Three-
mm-thick undecalcified sections. Original magnification 3 330. Bar 5 50 mm.
312
because it may be possible to optimize this protective
effect in future embedding protocols. (d) Methylbenzoate
acts as a plasticizer in MMA embedding mixtures.
Uniform polymerization of large MMA-infiltrated spec-
imens can be achieved only when the composition of
the last infiltration medium closely resembles the com-
position of the embedding mixture. Therefore, for the
method presented here it was necessary to change the
composition of the embedding medium to account for
the plasticizing effect of methylbenzoate. (e) The amount
of the accelerator N,N-dimethyl-p-toluidine used for
chemical polymerization at low temperatures has been
reduced in our method, resulting in a lower tendency
for bubble formation and more homogeneous poly-
merization of the blocks. (f) Our method does not re-
quire purification and destabilization of the methacry-
lates used for infiltration and polymerization.
High-quality undecalcified sections with few or no
artifacts are a prerequisite for reliable histomorpho-
metric measurements (Baron et al. 1983). The embed-
ding technique described here allows the preparation
of undecalcified sections with a histological quality
comparable to that of conventionally MMA-embed-
ded bone specimens and can therefore be routinely
used for bone histomorphometry. We have performed
histomorphometry on several hundred bones from
rats and other species embedded with this technique in
our laboratory. In our experience, the method can be
used without modification (except for altered dehy-
dration and infiltration times) for bones of rats, mice,
chicken, and cats, as well as for soft tissues. The method
may also be applicable to human iliac crest biopsies.
The enzyme activities of AP and TRAP were well
preserved by the embedding technique used in this
study. TRAP is a lysosomal enzyme expressed at high
levels in committed osteoclast precursors and mature
osteoclasts (Takahashi et al. 1994). It has recently
been shown that TRAP activity in human osteoclasts
is resistant to concentrations of tartrate of up to 150–
200 mM (Kadoya et al. 1994). At the concentration of
tartrate used in this study (100 mM), demonstration
of TRAP activity was restricted to osteoclasts and
mononuclear cells in close proximity to bone resorp-
tion sites in the rat. AP is an enzyme localized on the
external surface of the cell membrane of osteoblasts
and also odontoblasts (Tenorio et al. 1992; Doty and
Schofield, 1984). In agreement with the findings of the
present study, it is known that tissue AP activity is
very sensitive to fixation (Doty and Schofield 1984).
With the methods employed in this study, Kupffer
cells in the liver and osteoclasts were intensely labeled
by the MAb ED1. Proteolytic digestion was not neces-
sary for demonstration of positive staining with ED1.
In our experience, the rat antigen reacting with MAb
ED1 is resistant to most fixatives (e.g., ethanol, ace-
tone, PFA, formalin, Schaffer’s solution, and Carnoy’s
Erben
solution). The intense labeling of cells by immunohis-
tological techniques shown in this study is likely due
to the fact that complete removal of plastic is possible
in sections of MMA-embedded tissue (Baskin et al.
1992). The MAb ED1 recognizes a mainly intracellu-
larly localized 97-kD protein present in almost all cells
of the rat mononuclear phagocyte system, and ED1
can be used as a pan-macrophage marker in the rat
(Damoiseaux et al. 1994). It is known that rat osteo-
clasts also stain with MAb ED1 (Lee et al. 1995;
Sminia and Dijkstra 1986). The protein that is labeled
by MAb ED1 is mainly located on lysosomal mem-
branes and is probably homologous to human CD 68
(Damoiseaux et al. 1994). In frozen sections of bone
tissue, human osteoclasts were also shown to stain
with an MAb directed against human CD 68 (Kadoya
et al. 1994).
In summary, we have described a novel MMA em-
bedding technique that can be used for standard bone
histomorphometry and allows the application of his-
tochemical and immunohistochemical methods. There-
fore, this method meets the criteria for a more broad
application in bone research, and might be useful for
the inclusion of histochemical and immunohistologi-
cal methods in bone histomorphometry. In combina-
tion with histochemical or immunohistological meth-
ods specific for osteoblasts or osteoclasts, this embedding
method may also be useful as a basis for automatic de-
tection of these cells in undecalcified sections of bone
tissue by image analysis techniques. Moreover, the
embedding technique described here may represent a
further step towards the integration of different disci-
plines into bone research [“molecular histomorphom-
etry” (Parfitt 1994)], and may therefore provide new in-
sights into the physiology and pathology of bone tissue.
Acknowledgments
I thank Stefanie Engert for expert technical assistance.
Literature Cited
Baron R, Vignery A, Neff L, Silverglate A, Santa Maria A (1983)
Processing of undecalcified bone specimens for bone histomor-
phometry. In Recker RR, ed. Bone Histomorphometry: Tech-
niques and Interpretation. Boca Raton, FL, CRC Press, 13–35
Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Im-
provements of immunostaining of samples embedded in meth-
acrylate: localization of microtubules and other antigens through-
out the developing organs in plants of diverse taxa. Planta 187:
405–413
Britten KM, Howarth PH, Roche WR (1993) Immunohistochemis-
try on resin sections: a comparison of resin embedding tech-
niques for small mucosal biopsies. Biotech Histochem 68:271–
280
Damoiseaux JGMC, Döpp EA, Calame W, Chao D, MacPherson
GG, Dijkstra CD (1994) Rat macrophage lysosomal membrane
antigen recognized by monoclonal antibody ED1. Immunology
83:140–147
Doty SB, Schofield BH (1984) Ultrahistochemistry of calcified tis-
Methylmethacrylate Embedding Technique
sues. In Dickson GR, ed. Methods of Calcified Tissue Prepara-
tion. Amsterdam, Elsevier, 149–198
Frisch B, Lewis SM, Burkhardt R, Bartl R (1985) Biopsy Pathology
of Bone and Bone Marrow. London, Chapman and Hall
Kadoya Y, Al-Saffar N, Kobayashi A, Revell PA (1994) The expres-
sion of osteoclast markers on foreign body giant cells. Bone
Miner 27:85–96
Lee ER, Lamplugh L, Shepard NL, Mort JS (1995) The septoclast, a
cathepsin B-rich cell involved in the resorption of growth plate
cartilage. J Histochem Cytochem 43:525–536
Parfitt AM (1994) Osteonal and hemi-osteonal remodeling: the spa-
tial and temporal framework for signal traffic in adult human
bone. J Cell Biochem 55:273–286
Sato Y, Mukai K, Watanabe S, Goto M, Shimosato Y (1986) The
AMeX method. A simplified technique of tissue processing and
paraffin embedding with improved preservation of antigens for
immunostaining. Am J Pathol 125:431–435
Schenk RK, Olah AJ, Herrmann W (1984) Preparation of calcified
313
tissues for light microscopy. In Dickson GR, ed. Methods of Cal-
cified Tissue Preparation. Amsterdam, Elsevier, 1–56
Sminia T, Dijkstra CD (1986) The origin of osteoclasts: an immu-
nohistochemical study on macrophages and osteoclasts in embry-
onic rat bone. Calcif Tissue Int 39:263–266
Takahashi N, Udagawa N, Tanaka S, Murakami H, Owan I,
Tamura T, Suda T (1994) Postmitotic osteoclast precursors are
mononuclear cells which express macrophage-associated pheno-
types. Dev Biol 163:212–221
Tenorio D, Germain JP, Hughes FJ (1992) Histochemical studies of
acid and alkaline phosphatases in rat tooth germs with undecal-
cified resin-embedded specimens. J Histochem Cytochem 40:
1229–1233
Wolf E, Röser K, Hahn M, Welkerling H, Delling G (1992) Enzyme
and immunohistochemistry on undecalcified bone and bone mar-
row biopsies after embedding in plastic: a new embedding
method for routine application. Virchows Arch [A] 420:17–24