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Ž.
Comparative Biochemistry and Physiology Part A 129 2001 141᎐149
Surfactant proteins and cell markers in the respiratory
epithelium of the amphibian, Ambystoma mexicanum
Leigh-Anne D. Miller, Susan E. Wert, Jeffrey A. Whitsett
U
Children’s Hospital Medical Center, Di¨ision of Pulmonary Biology, Cincinnati, OH 45229-3039, USA
Received in revised form 15 December 2000; accepted 12 February 2001
Abstract
The respiratory tract is lined by diverse epithelial cell types whose morphology, gene expression and functions are
highly specialized along the cephalo-caudal axis of the lung. Pulmonary gas exchange, surface tension reduction, host
defense, fluid and electrolyte transport are functions shared by various vertebrate species, each organism facing similar
requirements for adaptation to air breathing. Consistent with this concept, we have identified distinct respiratory
epithelial cell populations in the amphibian, Ambystoma mexicanum, using morphologic, histochemical and im-
Ž.
munochemical techniques. Thyroid transcription factor-1 TTF-1 , a homeodomain nuclear transcription factor critical
Ž.
to lung formation, and surfactant protein B SP-B , an amphipathic polypeptide required for surfactant function, were
detected in the peripheral respiratory epithelial cells of the axolotl lung, in cells with characteristics of Type II alveolar
epithelial cells in mammals. -Tubulin and carbohydrate staining identified distinct subsets of ciliated and goblet cells.
SP-D, a member of the collectin family of innate host defense proteins, was also detected in peripheral epithelial cells of
the axolotl lung. Pulmonary surfactant and host defense proteins are shared across diverse phyla supporting the concept
that pulmonary structure and function have evolved from common ancestors. 䊚 2001 Elsevier Science Inc. All rights
reserved.
Keywords: Axolotl; Epithelium; Lung; SP-B; SP-D; TTF-1
1. Introduction
In most vertebrates the lung consists of con-
ducting airways leading to peripheral saccules,
wherein efficient gas exchange of CO and O
22
occurs from the airspaces to the pulmonary vascu-
U
Corresponding author. Children’s Hospital Medical Cen-
ter, Division of Neonatalogy and Pulmonary Biology, 3333
Burnet Avenue, Cincinnati, OH 45229-3039, USA. Tel.: q1-
513-636-4830; fax: q1-513-636-7868.
Ž.
E-mail address: jeff.whitsett@chmcc.org J.A. Whitsett .
lature. In vertebrates, the respiratory tract is lined
by various respiratory epithelial cells with charac-
teristic morphologic features. The cell types, gene
products, and functions vary developmentally and
spatially along the proximal to distal regions of
the airways. In the mammalian lung, the proximal
conducting airways are lined primarily by non-cili-
ated, secretory and ciliated cells, that play critical
roles in host defense, fluid and electrolyte
homeostasis and mucociliary clearance. Periph-
eral respiratory epithelial cells are adapted for
the synthesis and metabolism of surfactant pro-
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved.
Ž.
PII: S 1 0 9 5 - 6 4 3 3 0 1 00311-7
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L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149142
teins that are critical for surface tension reduc-
tion and innate host defense in the alveolar
spaces. Type II epithelial cells produce surfactant
lipids and proteins, while squamous Type I ep-
ithelial cells line most of the gas-exchange surface
of the alveoli.
The complex respiratory epithelium in the ma-
ture mammalian lung is established from progeni-
tor cells arising from an evagination of the foregut
endoderm that forms the primordial trachea and
lung buds. Transcriptional and autocrine᎐
paracrine signals influence the formation of the
mammalian lung and include the precise tem-
poral᎐spatial expression of transcription factors,
including the homeodomain protein, thyroid tran-
Ž.
scription factor TTF-1 , the winged helix protein,
HNF-3, and the zinc-finger protein, GATA-6.
These factors regulate expression of target genes,
including surfactant proteins and Clara cell secre-
tory proteins that are characteristic of developing
and mature alveolar, bronchial, and bronchiolar
Ž.
cell types Perl and Whitsett, 1999 . Ciliated cells
lining conducting airways are specified, at least in
part, by the actions of the forkhead homologue,
HFH-4, and are marked by the expression of cilia
Ž
specific genes, including -tubulin Tichelaar et
.
al., 1999 . Recent studies demonstrated signaling
pathways, both transcriptional and autocrine᎐
paracrine, that mediate pharyngeal development
in Caenorhabdites elegans and tracheal mor-
phogenesis in Drosophila, which are also shared
Ž.
in the mammalian lung Perl and Whitsett, 1999 .
Members of the FGF family of polypeptides are
critical to lung morphogenesis. Their effects are
mediated by FGF receptors, FGFRIIIb in particu-
lar, and are critical for formation of the lung buds
Ž
Peters et al., 1994; Min et al., 1998; Arman et al.,
.
1999 .
The amphibian lungs, consisting of two uni-
cameral lung sacs lined by diverse respiratory
epithelial cells, share many morphological fea-
tures with those in other vertebrates. As assessed
by morphologic and biochemical criteria, lungs of
both amphibians and mammals contain pulmo-
nary surfactant and are lined by cells that share
morphologic features of Type II epithelial cells
seen in the mammalian and avian lung. In mam-
mals and birds, these cells express TTF-1 and its
Ž
downstream targets, the surfactant proteins Zhou
.
et al., 1996; Zeng et al., 1998 .
The present study was undertaken to test
whether the amphibian, Ambystoma mexicanum,
expressed TTF-1, surfactant proteins and other
markers of the diverse respiratory epithelial cell
types characteristic of mammalian and avian
lungs.
2. Materials and methods
2.1. Animal maintenance
Ž.
Adult axolotls Ambystoma mexicanum used in
this study were raised from eggs obtained from
the axolotl colony at the University of Guelph,
Guelph, Canada. All animals were raised and
maintained in the laboratory using standard
methods of care and feeding in accordance with
the principles and guidelines of the Canadian
Council on Animal Care.
2.2. Lung histochemistry
Before embedding in paraffin, lung tissues were
obtained from axolotls anesthetized with MS-222,
fixed overnight at 4⬚C in 4% neutral buffered
paraformaldehyde in 0.1 M PBS, and washed in a
Ž.
graded series of ethanols. Sagittal sections 5-m
were mounted on poly-lysine-coated slides for
histochemistry and silane-coated slides for im-
munolabeling. Sections were deparaffinized and
rehydrated before staining with hematoxylin and
eosin or alcian blue.
Alcian blue staining for mucociliary substances
was carried out using 5-m sections that were
deparaffinized, rehydrated and incubated in 3%
aqueous acetic acid for 3 min. Slides were next
incubated in 1% alcian blue in 3% acetic acid
Ž.
pH 2.5 for 30 min and then washed in running
water for 10 min. After rinsing in distilled water,
the slides were counterstained in 0.1% nuclear
Ž.
fast red Kernechtrot for 5 min and washed for
another minute in running water. Before mount-
ing, the slides were dehydrated in 95% ethanol,
absolute ethanol and cleared in xylene.
2.3. Antibodies
A rabbit polyclonal antibody to rat TTF-1 was
generated to the soluble fraction of the thyroid
transcription factor protein, absorbed against ma-
ture liver cells and used at a concentration of
1:1000 on the axolotl lung. A mouse monoclonal
antibody to -tubulin IV was obtained from Bio-
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149 143
Ž.
Genex San Ramon, CA and was used on axolotl
lung sections at a concentration of 1:160. A rabbit
polyclonal antibody to the pro-protein of surfac-
Ž.
tant protein C proSP-C was generated and used
at dilutions of 1:1000, 1:2000, 1:3000 and 1:4000.
A rabbit polyclonal antibody to the mature sur-
Ž.
factant protein B SP-B peptide was isolated
from chloroform᎐methanol extracts of bovine
lung lavage and used at a concentration of 1:3000
on axolotl lung tissue. Rabbit polyclonal antibody
was generated to murine surfactant protein D
Ž.
SP-D and absorbed against SP-D null lungs. The
SP-D antibody was initially purified using a proce-
Ž.
dure previously described by Strong et al. 1998 .
Briefly, supernatant obtained by centrifugation of
lung lavage at 10 000=g for 40 min is applied to
a maltosyl-agarose column and the bound SP-D
specifically eluted using MnCl . The antibody was
2
further purified by gel-filtration on Superose-6.
Immunoblotting demonstrated that this antibody
recognizes a 42-kDa protein band and its
oligomers. Specificity of the TTF-1, proSP-C, SP-B
and -tubulin antibodies was established previ-
Ž
ously Bannerjee et al., 1992; Clark et al., 1995;
.
Vorbroker et al., 1995; Zhou et al., 1996 .
2.4. Immunohistochemistry
Immunohistochemical staining for TTF-1 was
carried out using 5-m paraffin sections that were
deparaffinized, rehydrated and heated up to 90⬚C
Ž
in 0.1 M citric acid in 0.1 M sodium citrate pH
.
6.0 for an initial 15 min and three successive
5-min periods. After cooling, the sections were
treated with 3% hydrogen peroxide in methanol
for 15 min, blocked in 2% goat serum in 0.1%
TBS for 2 h at room temperature, and incubated
in TTF-1 primary antibody at 4⬚C overnight. Con-
trol sections were incubated in blocking serum
alone at 4⬚C overnight. After application of pri-
mary antibody, sections were developed with a
biotinylated goat anti-rabbit secondary antibody
Ž
and a Vector Elite ABC kit Vector Laboratories;
.
Burlingame, CA . Application of NiDAB en-
hanced antigen localization and was followed by
incubation in Tris᎐cobalt and counterstaining
with nuclear fast red.
SP-B, proSP-C and SP-D immunolabeling
procedures were similar to those described above
consisting of deparaffinization and rehydration of
5-m sections, incubation in 3% hydrogen perox-
ide and methanol, blocking in 2% goat serum
with subsequent overnight incubation with the
primary antibodies. Control tissues were in-
cubated overnight at 4⬚C in blocking serum only.
Development of sections occurred as described
above. For -tubulin immunolabeling, sections
were blocked in 2% horse serum and then in-
cubated overnight at 4⬚C in the -tubulin primary
antibody. As described above, the sections were
then developed but using biotinylated horse anti-
mouse secondary antibody.
All immunohistochemical procedures were car-
ried out with negative controls lacking primary
antibody and positive controls consisting of mouse
lung tissue to ensure specificity of the staining.
3. Results
3.1. Histological obser
¨ations
Adult axolotl lung contains a central airspace
lined by alveolar folds, represented diagramati-
cally in Fig. 1. From sagittal sections, the alveolar
folds extend circumferentially along the lumen of
the lung and project into the central airspace
Ž
dividing it into smaller airpockets Figs. 1 and
.
2a,b . These alveolar folds consist of a large,
central blood vessel surrounded by smooth mus-
cle and covered by a thin epithelium that also
Ž.
lines the lumen of the lung Fig. 2b . The epithe-
lium consists of pneumocytes, ciliated and goblet
cells. Cilia were readily detectable even at low
Ž.
magnification Fig. 2b . The large blood vessels
contained nucleated blood cells. Numerous
smaller capillaries were located adjacent to the
epithelium lining the alveolar airpockets, in asso-
Ž.
ciation with pneumocytes Fig. 2b . Goblet cells
were readily detected using alcian blue to stain
Ž
the carbohydrates produced by these cells Fig.
.Ž.
2c . The outer adventitial layer Figs. 1 and 2a
contained smooth muscle, blood vessels, connec-
tive tissue and some small clusters of pigmented
Ž.
cells not shown .
3.2. Distribution of TTF-1 protein in adult axolotl
lung tissue
TTF-1 was detected throughout the axolotl lung
in subsets of cells of the epithelium lining the
Ž.
airpockets Fig. 3a,b . Distinct nuclear staining,
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149144
characteristic of TTF-1 immunolabeling in mam-
malian lung, was observed.
3.3. Distribution of

-tubulin IV in adult axolotl
lung tissue
Only ciliated cells from the epithelial layer of
the axolotl lung were labeled with -tubulin anti-
Ž.
body Fig. 3c,d . -Tubulin staining correlated
Ž.
with presence of cilia Fig. 3d confirming its
specificity for ciliated cells. Ciliated cells were
abundant and heterogeneously distributed
throughout the epithelium of the axolotl lung
Ž.
Fig. 3 .
3.4. Distribution of surfactant proteins in adult
axolotl lung tissue
Ž.
Immunostaining for SP-B Fig. 4a,b and SP-D
Ž.
Fig. 4c,d was detected in subsets of epithelial
cells lining the axolotl lung. Extracellular labeling
was observed for both proteins, indicating the
presence of SP-B and SP-D in the lumen of the
axolotl lung. In general, more cells expressed
SP-B than SP-D, a pattern of staining that was
consistent throughout the lungs. Specific im-
Ž.
munolabeling of SP-C not shown was not de-
Fig. 1. Diagram of general structure of adult axolotl lung.
Ž.
Fig. 2. Histology of adult axolotl lung. a Low magnification
view from sagittal sections of adult axolotl lung stained with
hematoxylin and eosin. Bracket indicates whole alveolar septa;
asterix indicates airpocket; thin arrows indicate epithelial
layer; thick arrows indicate smooth muscle; arrowheads indi-
Ž.
cate nucleated amphibian blood cells. Scale bars125 m. b
Ž.
High magnification view of section in a . Thick arrows indi-
cate ciliated cells; thin arrows indicate pneumocytes; large
arrowheads indicate goblet cells; small arrowheads indicate
Ž.
capillaries. Scale bar s50 m. c Low magnification view of
sagittal section from adult axolotl lung stained with alcian
Ž.
blue. Arrows indicate goblet cells stained bright blue . Scale
bars 125 m.
tected in the axolotl lungs, indicating that this
protein was not expressed or did not cross-react
with the antiserum that was generated against
human proSP-C.
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149 145
Ž.
Fig. 3. TTF-1 and -tubulin IV immunolabeling of adult axolotl lung. a Low magnification view of sagittal section of adult axolotl
Ž. Ž.
lung immunostained for TTF-1. Note dense nuclear staining of subsets of cells arrows . Scale bar s250 m. b High magnification of
Ž. Ž.
section in a . Arrows indicate nuclei of cells immunolabeled with TTF-1. Scale bar s 50 m. c Low magnification view of sagittal
Ž. Ž.
section of adult axolotl lung immunostained for -tubulin. Scale bar s 125 m. d High magnification view of section in c . Note that
-tubulin immunolabeling localizes to the cilia of ciliated cells. Scale bars 25 m.
4. Discussion
Our present study demonstrates remarkable
conservation of respiratory epithelial cell types
and proteins in the amphibian, Ambystoma mexi-
Ž.
canum axolotl , and mammals. Surfactant protein
B, a critical component of pulmonary surfactant,
and surfactant protein D, an important innate
host defense protein, were detected in the respi-
ratory epithelium of this salamander. The respira-
tory epithelium of the axolotl lung, like that in
the mammalian lung, consisted of diverse cell
types, including secretory, ciliated and cuboidal
cells that likely contribute to specific aspects of
lung function such as innate host defense and
surfactant homeostasis.
The axolotl lung is less complex than that of
mammals. The paired tubular structures are sac-
like and well vascularized containing a central
airspace penetrated by alveolar septa similar to
Ž.
those seen in the frog Okada et al., 1962 and
Ž
Amphiuma tridactylum Bell and Stark-Vancs,
.
1983 . In cross-section, the alveolar septa appear
as finger-like projections that contain a central
large blood vessel surrounded by smooth muscle
and covered with an epithelial layer that lines the
lumen of the air pockets. This epithelial layer is
non-uniform and composed of four cell types:
pneumocytes, ciliated cells, goblet cells, and neu-
roepithelial bodies. Pneumocytes form the inter-
nal surface of the blood᎐gas barrier and are
characterized by abundant osmiophilic lamellar
Ž.
bodies Scheuermann et al., 1989 indicative of
Ž
the presence of an axolotl surfactant Dierichs
.
and Dosche, 1982; Keyhani, 1987 . These cells
appear to combine the morphological characteris-
tics of mammalian Type I and Type II cells
Ž.
Scheuermann et al., 1989 and the latter are
likely the site of SP-B staining seen in cuboidal
cells in the present study. Both the ciliated cells,
which localize over the septa, and the goblet cells,
which are more dispersed, contain osmiophilic
intracellular bodies, although fewer than those
Ž
seen in the pneumocytes Dierichs and Dosche,
.
1982 . The abundant goblet cells that stained with
alcian blue in the axolotl lung suggest the produc-
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149146
Ž.
Fig. 4. SP-B and SP-D immunolabeling of adult axolotl lung. a Low magnification view of sagittal section of adult axolotl lung
Ž.
immunostained for SP-B. Only subsets of epithelial cells are immunolabeled with SP-B. Scale bar s 125 m. b High magnification
Ž. Ž.
view of section in a . Arrows indicate pneumocytes labeled with SP-B. Scale bar s25 m. c Low magnification view of sagittal section
Ž.
of adult axolotl lung immunolabeled with SP-D. Only subsets of epithelial are stained. Scale bar s50 m. d High magnification view
Ž.
of section in c . Arrows indicate pneumocytes immunolabeled with SP-D. Scale bar s25 m.
tion of mucins. While the presence of -tubulin
staining in subsets of respiratory epithelial cells is
consistent with the presence of ciliated cells that
are important in pulmonary innate defense and
mucociliary clearance. These two distinct cell
types, goblet cells and ciliated cells, were found in
divergent sites in the salamander, reminiscent of
Ž
those characterizing the mammalian lung Perl
.
and Whitsett, 1999 .
Compared to mammals and axolotls, the lungs
of frogs are simpler in structure, consisting of
simple bilateral, spindle-shaped sacs directly con-
Ž.
nected to the larynx Okada et al., 1962 . Cross-
sections of these sacs reveal a central airspace
Ž
penetrated circumferentially by septa Okada et
.
al., 1962 . The septa, consisting of blood vessels,
smooth muscle, lymphatics, and epithelium, di-
vide the central airspace into smaller pockets
Ž.
where gas exchange occurs Okada et al., 1962 .
These air-pockets are considered to be primitive
alveoli while the central undivided airspace, or
vorbronchus, is thought to be comparable to the
Ž.
mammalian bronchus Okada et al., 1962 . The
primitive alveolus is completely lined with epithe-
lium that consists of two cell types. One cell type
is similar to the small alveolar cell in mammalian
pulmonary epithelium, containing some osmio-
philic bodies and microvilli on the cell surface
Ž.
facing the airspace Okada et al., 1962 . The
other type is reminiscent of the mammalian large
alveolar epithelial cell with more osmiophilic
Ž.
bodies and fewer microvilli Okada et al., 1962 .
In mammals, a complex mixture of phospho-
lipids, neutral lipids and proteins called pulmo-
nary surfactant coats the inner surface of the lung
that prevents adhesion of pulmonary surfaces af-
ter exhalation. Three major lipid components of
pulmonary surfactant include disaturated phos-
Ž.
pholipids DSPs , unsaturated phospholipids
Ž.
USPs and cholesterol. DSPs act to lower the
surface tension of the fluid lining the lung while
cholesterol and USPs function to promote re-
spreading of pulmonary surfaces upon inflation of
the lung. Pulmonary surfactant has also been
found in various species of amphibians including,
Siren intermedia, Amphiuma tridactylum, Bufo
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149 147
marinus, Ambystoma tigrinum, and Ambystoma
mexicanum. Amphibian surfactant appears to be
secreted as osmiophilic lamellar bodies from spe-
cific secretory pneumocytes into the airspaces
Ž.
Pattle et al., 1977; Stark-Vancs et al., 1984 .
There appears to be at least two evolutionary
trends in vertebrate surfactant composition. First,
the total amount of surfactant lipids, as de-
termined from wet lung masses, generally in-
creases from very low levels in the fish, to inter-
mediate levels in amphibians reaching its highest
Ž.
levels in mammals Daniels et al., 1995 . Second,
the general composition of pulmonary surfactant
varies from high cholesterol with very low DSP in
primitive air-breathing fishes, to intermediate
cholesterol with intermediate DSP in amphibians,
to low cholesterol and high DSP in reptiles and
Ž.
mammals Daniels et al., 1995 . It was therefore
suggested that pulmonary surfactant consisting of
high cholesterol and low DSPs is a primitive
Ž.
surfactant or protosurfactant Daniels et al., 1995 .
Specifically, the lipid composition of surfactant in
Bufo marinus and other frogs was found to be
similar to that of mammals in containing pre-
Ž.
dominantly phosphatidylcholine PC with sub-
Ž.
stantial amounts of phosphatidylglycerol PG
Ž.
Daniels et al., 1994 . This profile differed from
that of the aquatic salamanders which did not
Ž.
contain PG but phosphatidylinositol PI and twice
Ž.
the amount of cholesterol Daniels et al., 1994 .
In mammalian surfactant, there are four pro-
tein components that have been described, sur-
factant proteins, SP-A, SP-B, SP-C and SP-D.
Ž.
Sullivan et al. 1998 suggested that the presence
of these proteins across the vertebrate groups
would support the possibility that the surfactant
system is homologous. Using Western blot analy-
sis, immunocytochemistry and northern blot anal-
Ž.
ysis, Sullivan et al. 1998 detected the presence
of SP-A in several vertebrate classes including,
Osteichthyes, Amphibia, Reptilia, Aves and
Mammalia. Another surfactant protein, SP-B has
been detected in the non-mammalian vertebrates
Ž.
such as the chick Zeng et al., 1998 and the
Ž.
freshwater turtle Johnston et al., 2000 .
While the function of pulmonary surfactant is
unknown in amphibians, it has been proposed to
act similarly to mammalian surfactant in reducing
surface tension to prevent adhesion of pulmonary
surfaces and subsequent alveolar collapse
Ž.
Daniels et al., 1994; Orgeig et al., 1994 . Pulmo-
nary surfactant in mammals also acts as an anti-
edema agent and has been proposed to perform a
similar function in non-mammalian vertebrates
Ž.
Daniels et al., 1995 . The higher cholesterol con-
tent of surfactant in aquatic salamanders may
enable it to be functional at low body tempera-
tures, at which mammalian surfactant would be
Ž.
inactive Daniels et al., 1994; Orgeig et al., 1994 .
Ž.
Daniels et al. 1995 further proposed that pulmo-
nary surfactant in non-mammalian vertebrates
may aid the mucociliary escalator in the removal
of inhaled foreign particles.
From our observations and previous morpho-
logic studies, it appears that pulmonary structure
has been conserved between amphibians and
mammals. Respiratory surfaces, including skin and
lungs, require a liquid᎐air interface for oxygen
absorption. Therefore, it was expected that the
mechanism that evolved in mammals, to reduce
the high surface tension of fluid, would be con-
served in amphibians. Our study demonstrates
strong cross reactivity of both mammalian SP-B
and SP-D antibodies with the axolotl lung. SP-B
is a highly conserved amphipathic peptide synthe-
sized exclusively by Type II epithelial cells in
mammals. Lack of SP-B results in respiratory
failure in human infants and SP-B targeted mice
Ž.
Nogee et al., 1994; Clark et al., 1995 . Type II
cells in SP-B gene-targeted animals lacked lamel-
Ž.
lar bodies and tubular myelin Clark et al., 1995 .
In the mammalian lung, SP-D plays important
roles in the regulation of surfactant lipid concen-
trations and innate host defense mechanisms
Ž
Botas et al., 1998; Korfhagen et al., 1998; Law-
.
son and Reid, 2000 .
In summary, pulmonary cells from the amphib-
ian, Ambystoma mexicanum, expressed the surfac-
tant proteins B and D, as well as TTF-1, a critical
regulator of lung morphogenesis and surfactant
Ž
protein gene expression in mammals Bohinski et
.
al., 1994; Kimura et al., 1996 . The diversity of
ciliated and secretory cells characteristic of mam-
malian and avian lungs are well conserved in this
amphibian.
Acknowledgements
We thank Dr James Bogart for providing the
axolotls for this study and Ms Tana McDaniel for
preparation of axolotl lung tissue for paraffin
embedding and sectioning. We also thank Ms
Sherri Profitt for excellent technical assistance
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141᎐149148
with immunohistochemistry. This study was sup-
ported by funding from NIH grant SCOR
HL56387.
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