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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).


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.



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.



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.



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.



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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methods. Berlin: Springer-Verlag; 2010.2 Zou J, Luo H, Zeng Q, Dong Z, Wu D, Liu L. Protein kinase

CK2alpha is overexpressed in colorectal cancer and modulatescell proliferation and invasion via regulating EMT-relatedgenes. J Transl Med. 2011;9:97.

3 Lin KY, Tai C, Hsu JC, Li CF, Fang CL, Lai HC, et al.Overexpression of nuclear protein kinase CK2 alpha catalyticsubunit (CK2alpha) as a poor prognosticator in humancolorectal cancer. PLoS One. 2011;6:e17193.

4 Landesman-Bollag E, Romieu-Mourez R, Song DH, Sonen-shein GE, Cardiff RD, Seldin DC. Protein kinase CK2 inmammary gland tumorigenesis. Oncogene. 2001;20:3247–57.

5 Rodier G, Montagnoli A, Di Marcotullio L, Coulombe P,Draetta GF, Pagano M, et al. p27 cytoplasmic localization isregulated by phosphorylation on Ser10 and is not a prerequisitefor its proteolysis. EMBO J. 2001;20:6672–82.

6 Esposito V, Baldi A, De Luca A, Groger AM, Loda M,Giordano GG, et al. Prognostic role of the cyclin-dependentkinase inhibitor p27 in non-small cell lung cancer. Cancer Res.1997;57:3381–5.

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8 Tomasello G, Sciume C, Rappa F, Rodolico V, Zerilli M,Martorana A, et al. Hsp 10, Hsp70, and Hsp90 immunohisto-chemical levels change in ulcerative colitis after therapy. Eur JHistochem. 2011;55:e38.

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