Technical Note Immunohistochemistry on decalcified rat ... · Technical Note Immunohistochemistry...
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Technical Note
Immunohistochemistry on decalcified ratnasal cavity: trials and successes
Nancy Harris1, Charleata A. Carter2, Manoj Misra2, Robert Maronpot1
1Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, USA, 2Lorillard, Inc., Greensboro, NC,USA
Immunohistochemical (IHC) staining for Casein Kinase 2 alpha (CK2a), cyclin-dependent kinase inhibitor1B (p27Kip1), and heat shock protein 90 (HSP90) was performed on four levels of decalcified rat nasalcavity from four male and three female controls following nose-only exposure to ambient air for 5 days.Successful staining with negligible background artifact was obtained on multiple epithelial andneuroepithelial soft tissues, without antigen retrieval. Independent optimization was needed for each ofthe three antibodies. A major challenge in this project was maintaining tissue adherence to the slidesduring tissue incubations. This report documents immunolocalization of CK2a, p27Kip1, and HSP90 inrespiratory and olfactory tissue of the rat nose.
Keywords: CK2a, Decalcification, HSP90, Immunohistochemistry, Olfactory epithelium, p27Kip1, Rat nasal tissue, Respiratory epithelium
IntroductionThe challenges associated with obtaining high quality
immunohistochemistry (IHC) include variables such
as tissue fixation, antibody specificity, antigen retrie-
val, reagent dilution, and incubation times.1
Additional challenges are introduced in performing
IHC on rodent noses that were previously archived
and subsequently decalcified. Decalcified rodent nose
is difficult to section because the incisor and molar
teeth retain a degree of hardness and easily develop
tissue folds. The bony tissue structures have trouble
adhering to the glass slide and often lift off during the
numerous incubation steps of the staining process.
Such is the case in performing IHC on decalcified
sections of the noses of rats and mice. Nasal sections
from small rodents contain a range of tissue types,
from delicate ciliated respiratory epithelium to
cartilage to dental enamel. These animals are used
in testing drugs, pesticides, water disinfection by-
products, and other chemicals involved in environ-
mental exposures in order to identify potential
hazards to human health.
High throughput microarray and proteomic tech-
nologies generate hundreds to thousands of expression
profiles. In analyzing these data it is most practical to
initially focus on changes most clearly up- or down-
regulated. Three immunohistochemical assays, CK2a,
p27Kip1, and HSP90, were selected based on the most
promising data obtained from proteomic analysis of
nose tissue lysates in tobacco smoke-exposed versus
control rats. Since the proteomic data from the smoke-
exposed rats did not appropriately confirm the IHC for
the selected proteins, the study was restricted to controls.
CK2a is a highly conserved serine/threonine kinase
that phosphorylates substrates involved in a wide
variety of biological and pathological processes.2–4 It
shows both cytoplasmic and nuclear localization by
IHC and plays a critical role in the cell cycle,
influencing cell proliferation and cellular senescence.4
P27Kip1, a member of the kinase inhibitory protein
family, functions as a critical switch in growth arrest,
prompting subsequent cellular differentiation or
apoptosis.5,6 It, too, can be localized in both the
nucleus and the cytoplasm.5 Finally, HSP90 is highly
abundant in eukaryotic organisms, accounting for 1–
2% of normal cellular protein.7 It is involved in
folding, translocation, and degradation of intracel-
lular protein in both normal and stress conditions, is
induced by environmental stress, is involved in
oxidative stress defense, and can trigger innate and
adaptive immunity.8
The purposes of this paper are to present efforts to
obtain useful IHC on air-exposed control rats from
Correspondence to: Nancy Harris, Experimental Pathology Laboratories,Inc., PO Box 12766, Research Triangle Park, NC 27709 919-998-9407.Email: [email protected]
One continuing education contact hour can be earned by reading thisarticle and taking a short test; for details see www.nsh.org
92� National Society for Histotechnology 2013DOI 10.1179/2046023613Y.0000000027 Journal of Histotechnology 2013 VOL. 36 NO. 3
this study and to show some of the results in the
different cell types of the nose.
Materials and MethodsAnimal exposureThe study protocol was approved by the Institutional
Animal Care and Use Committee at the Illinois
Institute of Technology Research Institute. Male and
female Fischer rats, numbering 344, at 5 weeks of age
were exposed to filtered air to serve as controls for a
smoke inhalation study. Nose-only exposures were
for 3 hours/day for 5 consecutive days. Scheduled
necropsies were conducted on study day 5, immedi-
ately after final exposure.
Tissue processing/histopathological evaluationsHeads were collected from all animals after the
mandibles and skin were removed. Noses from five
control rats of each sex were infused with and
immersed in neutral buffered formalin (NBF) for
24 hours and then placed in 70% ethanol. Fixed
tissue in 70% ethanol was archived for several years.
Since tissues from three heads were dessicated based on
visual examination, the study was limited to the seven
archival samples (four males and three females) that
were well preserved without evidence of dehydration.
Noses from two males were decalcified in 14%
ethylenediaminetetraacetic acid (EDTA) for 18 days.
All other noses were decalcified with ImmunoCalTM
Figure 1 (A) Hematoxylin & eosin (H&E). Rat 1M. Ciliated respiratory epithelium lining the septum in the anterior nasal cavity
of a Sprague-Dawley control rat. The respiratory epithelium consists of tall columnar goblet cells (arrows) and basal cells
(filled arrowheads). Submucosal glands (open arrowhead) and their glandular ducts (asterisks) are adjacent to the underlying
cartilage. (B) CK2a. Rat 1M. Minimal immunopositivity is present in occasional basal cells (filled arrowhead) of the ciliated
respiratory epithelium and in apical cytoplasm of a submucosal gland duct (asterisk). (C) P27Kip1. Rat 2M. Moderate
immunostaining is present in glandular ducts in the submucosa, especially prominent in the apical cytoplasm (asterisks). Mild
punctate immunopositivity is present in the submucosal glands (open arrowhead) and in basal cells of the luminal epithelium
(filled arrowheads). (D) HSP90. Rat 2M. Marked HSP90 immunostaining is present in basal cells (filled arrowheads), with mild
immunopositivity in glandular ducts (asterisk) and minimal punctate positivity in submucosal glands.
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
Journal of Histotechnology 2013 VOL. 36 NO. 3 93
(Decal Chemical Corp., Tallman, NY, USA) for
6 days. Following decalcification, tissues were rinsed
in tap water, cross-sectioned at four levels, and
processed for routine paraffin embedding.9 Four
micron serial sections were mounted on poly-L-lysine
glass slides (Mercedes Medical Starfrost Adhesive
Slides, Sarasota, FL, USA) for hematoxylin & eosin
(H&E) and IHC-staining.
ImmunohistochemistryThe IHC staining for CK2a, p27Kip1, and HSP90
involved use of an automated stainer (DAKO Auto-
stainer, Carpinteria, CA, USA) to provide sequential
exposure of deparaffinized slides to 3% hydrogen
peroxide (Fisher Scientific, Pittsburgh, PA, USA),
avidin/biotin (Vector Laboratories, Burlingame, CA,
USA), and serum-free protein (DAKO, Carpinteria,
CA, USA) blocks followed by application of primary
antibody. Sources for primary antibodies were as
follows: CK2a (Anti-Casein Kinase 2a, clone 1AD9,
Millipore, Temecula, CA, USA), p27Kip1 (C-19, Santa
Cruz Biotechnology, Inc, Dallas, TX, USA), and
HSP90 (StressMarq, Victoria, BC, Canada). The incu-
bation time and dilution were based on assay optimiza-
tion studies using selected preliminary sections.
Following application of secondary antibody, the
Figure 2 (A) H&E. Rat 1M. Microanatomical features of the vomeronasal organ include bipolar neurons of the sensory
epithelium (arrows), supporting cells (filled arrowheads) of the sensory epithelium, pseudostratified epithelium (open
arrowheads), and glands (asterisk). The nares are lined by stratified squamous epithelium (large arrow). (B) CK2a. Rat 1M. Mild
to moderate immunopositivity is present in the pseudostratified epithelium (open arrowheads) and moderate punctate
immunostaining is present in the glands (asterisk). There is a thin line of immunopositivity on the surface of the sensory
epithelium. (C) P27Kip1. Rat 2M. There is moderate immunopositivity of the pseudostratified epithelium (open arrowhead) and
minimal immunopositivity of the glands (asterisk). The stratified squamous epithelium (large arrow) is strongly positive for
p27Kip1. (D) HSP90. Rat 1M. Marked immunostaining is present in the bipolar neurons of the sensory epithelium (arrow), the
pseudostratified epithelium (open arrowheads), and the stratified squamous epithelium of the nares (large arrow), with
moderate immunopositivity of the glands (asterisk).
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
94 Journal of Histotechnology 2013 VOL. 36 NO. 3
ABC label complex (Vector Laboratories, Bur-
lingame, CA, USA) was applied as per kit instructions
with DAB (Biocare Medical, Concord, CA, USA) as
the chromogen. Stained slides were counterstained with
hematoxylin.
CK2a immunostainingFor CK2a staining, the positive tissue type control
used was rat intestine. The negative isotype control
was purified mouse IgG, with a matching immuno-
globulin concentration to that of the primary anti-
body. Initial studies were performed without antigen
retrieval and with primary antibody dilutions of
1:100, 1:200, and 1:500 for an incubation time of
60 minutes. The resultant staining was weak. A
second trial using 1:100 and 1:500 dilution after a
heat retrieval step in citrate buffer, pH 6, for
45 minutes at 70uC provided too much background
staining. In a final trial, using a 1:100 (10 mg/ml)
primary antibody dilution without antigen retrieval,
slides were incubated for 90 and 120 minutes.
P27Kip1 immunostainingThe positive control for p27Kip1 was rat spleen. Tris-
buffered saline served as the negative control. Initial
dilutions of primary antibody used were 1:25, 1:50,
and 1:100 for 60 minutes incubation time without
antigen retrieval. Additional slides were treated with
a 1:50 primary dilution after heat retrieval in citrate
buffer, pH 6, for 60 minutes at 70uC. Staining was
very strong in the 1:25 dilution and the 1:50 with
heat-induced epitope retrieval, moderate for the 1:50
without antigen retrieval, and weak for the 1:100
dilution. The final successful trial used a 1:80 (2.5 mg/
ml) primary dilution for 60 minutes without antigen
retrieval.
Figure 3 (A) H&E. Rat 1M. Non-keratinized multilayered squamous epithelium (arrows) lines the nasolacrimal duct that drains
into the nasal cavity. (B) CK2a. Rat 1M. Minimal to mild cytoplasmic immunostaining is present in the multilayered epithelium
of the nasolacrimal duct, with occasional nuclear staining (arrows). (C) P27Kip1. Rat 1M. Mild cytoplasmic and moderate
nuclear immunopositivity is present in the nasolacrimal duct epithelium. (D) HSP90. Rat 1M. There is marked cytoplasmic
immunostaining for HSP90 in the nasolacrimal duct epithelium.
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
Journal of Histotechnology 2013 VOL. 36 NO. 3 95
HSP90For Hsp90 staining, the positive control was rat heart.
The negative isotype control was purified mouse
immunoglobulin G (IgG). Initial dilutions of primary
antibody were 1:40, 1:100, and 1:200 for 60 minutes
incubation time without antigen retrieval. The staining
was too strong across all three dilutions. In an attempt
for better IHC staining, the primary antibody was
diluted to 1:400, 1:800, 1:1000, and 1:1200. Also, the
secondary antibody was diluted from the initial 1:300
dilution to 1:400. Staining was too dark in the 1:400
and 1:800 dilutions of primary antibody, but began to
improve with the final selected 1:1200 (0.83 mg/ml)
dilution.
Specific staining details are provided in supple-
mentary material (Supplementary Materials 1–3
http://dx.doi.org/10.1179/2046023613Y.0000000027.S1–S3).
IHC evaluation
Scoring of IHC was based on the extent and intensity of
staining in the various major tissues lining the nasal
cavity. Subjective scores were as follows: 0 – no IHC
staining present; 1 – minimal IHC positivity; 2 – mild
IHC positivity; 3 – moderate IHC positivity; and 4 –
marked IHC positivity.
Figure 4 (A) H&E. Rat 1M. The olfactory epithelium in the posterior nasal cavity of a Sprague-Dawley rat consists of
sustentacular cells (arrows), olfactory sensory neurons (osn), basal cells (filled arrowheads), Bowman’s glands (asterisks),
and nerve bundles (n) in the lamina propria. (B) CK2a. Rat 4M. Mild immunopositivity is present in the cytoplasm of Bowman’s
glands. There is minimal positive staining in the olfactory sensory neurons and sustentacular cells. (C) P27Kip1. Rat 1M.
There is moderate immunopositivity for p27Kip1 in the cytoplasm and nuclei of Bowman’s glands as well as a positive
response in some of the sustentacular cell nuclei (arrows) and some of the olfactory sensory neurons in the mucosa. There is
light staining of the nerve bundles. (D) HSP90. Rat 2M. The olfactory mucosa and submucosa are strongly immunopositive for
HSP90, with the exception of the sustentacular cell nuclei (arrows). The staining reaction in Bowman’s gland is limited to the
cytoplasm, while the staining reaction in the olfactory sensory neurons is both cytoplasmic and nuclear. There is mild to
moderate staining of the nerve bundles.
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
96 Journal of Histotechnology 2013 VOL. 36 NO. 3
Tab
le2
P27K
ip1
imm
un
oh
isto
ch
em
istr
yin
nasal
cavit
ies
of
rats
exp
osed
by
no
se
on
lyto
filt
ere
dair
for
5co
nsecu
tive
days
Rat
#
Resp
irato
rym
uco
sa
an
dsu
bm
uco
sa
Olfacto
rym
uco
sa
an
dsu
bm
uco
sa
Vo
mero
nasal
org
an
Naso
lacri
mal
du
ct
Str
ati
fied
sq
uam
ou
sep
ith
eliu
m
Cili
ate
dre
sp
irato
ryep
ith
eliu
mS
ep
tal
su
bm
uco
sal
gla
nd
s
Bo
wm
an
’sg
lan
ds
Olfacto
rysen
so
ryn
eu
ron
sS
uste
nta
cu
lar
cells
Sen
so
ryep
ith
eliu
mP
seu
do
str
ati
fied
ep
ith
eliu
mV
om
ero
nasal
gla
nd
sC
yto
Nu
cC
yto
Nu
cC
yto
Nu
c
1M
*2
02
30
12
01
11
33
2M
*2
02
30
12
02
11
23
3M
20
33
12
11
21
12
34M
20
23
02
12
32
23
312F
20
43
03
12
42
23
313F
20
24
03
23
43
22
414F
20
24
13
22
31
23
4
All
imm
unosta
inin
gis
cyto
pla
sm
ic(C
yto
)unle
ss
ind
icate
dth
at
sta
inin
gis
nucle
ar
(Nuc).
Score
s:
05
no
IHC
sta
inin
g,
1z
5m
inim
al,
2z
5m
ild,
3z
5m
od
era
te,
4z
5m
ark
ed
,C
yto
–cyto
pla
sm
ic,
Nuc
–nucle
ar.
*D
ecalc
ifie
din
ED
TA
for
18
hours
.A
lloth
er
sam
ple
sd
ecalc
ifie
din
Imm
unoC
alT
M.
Tab
le1
CK
2a
imm
un
oh
isto
ch
em
istr
yin
nasal
cavit
ies
of
rats
exp
osed
by
no
se
on
lyto
filt
ere
dair
for
5co
nsecu
tive
days
Rat
#
Resp
irato
rym
uco
sa
an
dsu
bm
uco
sa
Olfacto
rym
uco
sa
an
dsu
bm
uco
sa
Vo
mero
nasal
org
an
Cili
ate
dre
sp
irato
ryep
ith
eliu
mS
ep
tal
su
bm
uco
sal
gla
nd
s
Bo
wm
an
’sg
lan
ds
Olfacto
rysen
so
ryn
eu
ron
sS
uste
nta
cu
lar
cells
Sen
so
ryep
ith
eliu
mP
seu
do
str
ati
fied
ep
ith
eliu
mV
om
ero
nasal
gla
nd
sN
aso
lacri
mal
du
ct
Str
ati
fied
sq
uam
ou
sep
ith
eliu
mC
yto
Nu
cC
yto
Nu
c
1M
*1
12
12
11
22
31
12M
*0
22
22
11
02
31
23M
11
32
11
22
33
11
4M
11
12
11
21
32
21
12F
11
22
12
12
33
22
13F
00
12
11
12
33
10
14F
11
12
12
21
22
11
All
imm
unosta
inin
gis
cyto
pla
sm
ic(C
yto
)unle
ss
ind
icate
dth
at
sta
inin
gis
nucle
ar
(Nuc).
Score
s:
05
no
IHC
sta
inin
g,
1z
5m
inim
al,
2z
5m
ild,
3z
5m
od
era
te,
4z
5m
ark
ed
,C
yto
–cyto
pla
sm
ic,
Nuc
–nucle
ar.
*D
ecalc
ifie
din
ED
TA
for
18
hours
.A
lloth
er
sam
ple
sd
ecalc
ifie
din
Imm
unoC
alT
M.
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
Journal of Histotechnology 2013 VOL. 36 NO. 3 97
ResultsHematoxylin & eosin stainingThere was good representation of the major tissue
components in H&E-stained sections from the four
cross-sectional levels of the nasal cavity. Tissues
included were: stratified squamous epithelium, vome-
ronasal organ, ciliated and non-ciliated respiratory
epithelium, submucosal glands, nasolacrimal duct,
and olfactory neuroepithelium with associated
Bowman’s glands. Although not evaluated by IHC,
the integrity and morphological features of nasal
turbinate bones and rodent incisor and molar teeth
were indicative of adequate decalcification of the rat
noses. For purposes of demonstration, photomicro-
graphs of respiratory epithelium of the nasal septum
(Fig. 1A), structures of the vomeronasal organ
(Fig. 2A), nasolacrimal duct (Fig. 3A), and olfactory
neuroepithelium (Fig. 4A) are provided.
ImmunostainingGood specific immunostaining for CK2a, without
objectionable background staining, was obtained
with the 120-minute incubation time at 1:100
(10 mg/ml) dilution (Figs. 1B, 2B, 3B, and 4B). No
background staining occurred in the negative isotype
control. IHC scores for different nasal tissue compo-
nents are presented in Table 1.
For p27Kip1, good differentiation of cellular com-
ponents was achieved by using a 1:80 (2.5 mg/ml)
primary dilution for 60 minutes (Figs. 1C, 2C, 3C,
and 4C). IHC scores for the different nasal tissue
components are presented in Table 2.
The 1:1200 (0.83 mg/ml) primary dilution presented
strong immunostaining for Hsp90 without excess
background (Figs. 1D, 2D, 3D, and 4D). No specific
staining was apparent in the negative control. IHC
scores for the different nasal components are presented
in Table 3.
DiscussionBased on evaluation of the H&E-stained sections, all
components of the nasal cavities of the seven rats
were within normal limits. As is typical for sections of
rodent noses, exact tissue comparison between animals
for each sampled level is not perfect due to normal
variation in tissue trimming following decalcification.
For purposes of illustrating results, photomicrographs
(Figs. 1–4) were obtained from the same rat whenever
possible. All photomicrographs are from male rats.
Acceptable IHC results on delicate soft tissues were
successfully obtained on these archival rodent tissues
following decalcification of the surrounding bony
structures of the nose. Tissues had been immersion-
fixed in NBF for 24 hours, then transferred to andTab
le3
HS
P90
imm
un
oh
isto
ch
em
istr
yin
nasal
cavit
ies
of
rats
exp
osed
by
no
se
on
lyto
filt
ere
dair
for
5co
nsecu
tive
days
Rat
#
Resp
irato
rym
uco
sa
an
dsu
bm
uco
sa
Olfacto
rym
uco
sa
an
dsu
bm
uco
sa
Vo
mero
nasal
org
an
Naso
lacri
mal
du
ct
Str
ati
fied
sq
uam
ou
sep
ith
eliu
m
Cili
ate
dre
sp
irato
ryep
ith
eliu
mS
ep
tal
su
bm
uco
sal
gla
nd
s
Bo
wm
an
’sg
lan
ds
Olfacto
rysen
so
ryn
eu
ron
sS
uste
nta
cu
lar
cells
Sen
so
ryep
ith
eliu
mP
seu
do
str
ati
fied
ep
ith
eliu
mV
om
ero
nasal
gla
nd
sC
yto
Nu
cC
yto
Nu
cC
yto
Nu
c
1M
*4
01
20
20
34
14
03
2M
*4
01
20
20
44
24
02
3M
40
11
03
04
43
30
14M
40
21
04
04
42
30
312F
40
12
03
14
42
40
213F
40
12
03
04
42
30
214F
40
12
03
04
41
30
2
All
imm
unosta
inin
gis
cyto
pla
sm
ic(C
yto
)unle
ss
ind
icate
dth
at
sta
inin
gis
nucle
ar
(Nuc).
Score
s:
05
no
IHC
sta
inin
g,
1z
5m
inim
al,
2z
5m
ild,
3z
5m
od
era
te,
4z
5m
ark
ed
,C
yto
–cyto
pla
sm
ic,
Nuc
–nucle
ar.
*D
ecalc
ifie
din
ED
TA
for
18
hours
.A
lloth
er
sam
ple
sd
ecalc
ifie
din
Imm
unoC
alT
M.
Harris et al. Immunohistochemistry on decalcified rat nasal cavity
98 Journal of Histotechnology 2013 VOL. 36 NO. 3
stored in 70% ethanol prior to decalcification and
staining. Each of the three IHC markers, Ck2a,
p27Kip1, and HSP90, required independent optimi-
zation of staining parameters to obtain good specific
immunopositivity in the variety of epithelial soft
tissues in the nose while ensuring negligible back-
ground staining. Antigen retrieval was not necessary
for successful immunostaining, although it could be
argued that exposure to the decalcification process
may represent a form of antigen retrieval. It was
noted that staining reactions within cartilage and
decalcified bone were variable within and between
tissue sections, even on the same slide. This may be a
reflection of differential degrees of decalcification of
hard tissue or, alternatively, may represent a non-
specific reaction in the biochemical composition of
cartilage and decalcified bone. Semi-quantitative
evaluation of staining showed similar results between
the two decalcification procedures, using either
EDTA or ImmunoCalTM
.
While optimizing the Ck2a, p27Kip1, and HSP90,
the goal was to obtain a balanced reaction of good
immunostaining between the different cellular com-
ponents of the nasal cavity. The strongest staining
was obtained for HSP90, consistent with its high
abundance in eukaryotic tissues.7 While staining
optimization for HSP90 yielded acceptable results
for all tissue components in the noses of the rats, addi-
tional optimization for vomeronasal organ (Fig. 2D)
and olfactory epithelium (Fig. 4D) might have im-
proved staining for these two tissue components.
A major challenge during staining was maintaining
tissue adherence to the slides during tissue incuba-
tion; bony tissue tended to lift from the slide during
processing, necessitating cutting additional sections for
repeat staining. During early stain optimization, a more
gentle method of antigen retrieval was chosen without
high pressure or boiling solutions in an effort to reduce
this problem. Ultimately, antigen retrieval was not
needed for the favorably balanced IHC staining among
the different cellular components in the rat nose.
Sectioning nasal turbinate blocks at 4 microns, allowing
the slides to air dry several days at room temperature
prior to staining, and avoiding antigen retrieval steps
when possible mitigated loss of tissue.
ConclusionAntigen retrieval, although necessary for many
antibodies, can introduce new challenges to the IHC
process. This study using CK2a, p27Kip1, and
HSP90 staining showed discrete staining with each
antibody in different cell types in the rat nose. The
ability to effectively stain for these proteins should
aid in predicting the effects of potential toxins on cell
proliferation, senescence, differentiation, apoptosis,
stress response, and protein folding in specific nasal
cell types.
AcknowledgementsMaterial presented in this paper is from a study
funded by Lorillard, Inc., Greensboro, NC, USA.
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