research article development of pathological diagnostics...

Research ArticleDevelopment of Pathological Diagnostics of Human KidneyCancer by Multiple Staining Using New Fluorescent Fluolid Dyes

Dilibaier Wuxiuer1 Yun Zhu1 Takunori Ogaeri1 Keiji Mizuki2 Yuki Kashiwa3 KentaroNishi3 Shin-ichiro Isobe3 Tei-ichiro Aoyagi4 and Ryoiti Kiyama1

1 Biomedical Research Institute National Institute of Advanced Industrial Science and Technology (AIST) 1-1-1 Higashi TsukubaIbaraki 305-8566 Japan

2Department of Nanoscience Faculty of Engineering Sojo University 4-22-1 Ikeda Kumamoto 860-0082 Japan3Department of Applied Chemistry and Biochemistry Faculty of Engineering Kyushu Sangyo University 2-3-1 Matsukadai Higashi-ku Fukuoka 813-8503 Japan

4 Ibaraki Medical Center Tokyo Medical University 3-20-1 Ami Inashiki Ibaraki 300-0395 Japan

Correspondence should be addressed to Ryoiti Kiyama kiyamaraistgojp

Received 16 February 2014 Accepted 20 May 2014 Published 3 June 2014

Academic Editor Paul Crispen

Copyright copy 2014 Dilibaier Wuxiuer et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Newfluorescent Fluolid dyes have advantages over others such as stability against heat dryness and excess lightHerewe performedsimultaneous immunostaining of renal tumors clear cell renal cell carcinoma (RCC) papillary RCC chromophobe RCC acquiredcystic disease-associated RCC (ACD-RCC) and renal angiomyolipoma (AML) with primary antibodies against Kank1 cytokeratin7 (CK7) and CD10 which were detected with secondary antibodies labeled with Fluolid-Orange Fluolid-Green and Alexa Fluor647 respectively Kank1 was stained in normal renal tubules papillary RCC and ACD-RCC and weakly or negatively in all othertumors CK7 was positive in normal renal tubules papillary RCC and ACD-RCC In contrast CD10 was expressed in renaltubules and clear cell RCC papillary RCC AML and AC-RCC and weakly in chromophobe RCC These results may contributeto differentiating renal tumors and subtypes of RCCs We also examined the stability of fluorescence and found that fluorescentimages of Fluolid dyes were identical between a tissue section and the same section after it was stored for almost three years at roomtemperature This indicates that tissue sections can be stored at room temperature for a relatively long time after they are stainedwith multiple fluorescent markers which could open a door for pathological diagnostics

1 Introduction

Owing to the increased availability of diagnostic markers forpathological evaluation of cancer there has been an increaseddemand for staining valuable specimens with multiple andcombinational markers There have been approaches basedon double triple and even quadruple staining of specimenswith the respective numbers ofmarkers [1ndash5] However therehas been difficulty in putting such staining methods intopractice due to various problems such as the quality ofmethods and the stability and biological relevance ofmarkers[6] When colorimetric staining is used such as that withalkaline phosphatase- or horseradish peroxidase-conjugatedantibodies multiplemarkers are hard to differentiate visually

In contrast when multiple fluorescent markers are used forstaining stained specimens cannot be stored for a long timedue to the poor stability of fluorescent dyes Thus a systemformultiple staining using stable fluorescent dyes is crucial todevelop a new diagnostic protocol for the pathological exam-ination of cancer A pathological application was exploredpreviously with a new fluorescent dye Fluolid-Orange [7]Another Fluolid dye Fluolid-Green is now available andthese Fluolid dyes show strong fluorescence even in the solidstate large Stokes shifts and stability against dryness heatand excess light [8] and are thus ideal for long-term storageof stained specimens

Kidney and urinary tract cancers accounted for 8334deaths in 2012 in Japan roughly 2 of all cancers [9]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 437871 6 pageshttpdxdoiorg1011552014437871

2 BioMed Research International

Renal cell carcinoma (RCC) is the most common type ofkidney cancer and is classified into five histologic subtypesclear cell (70ndash80) papillary (10ndash15) chromophobe (3ndash5) collecting duct (1) and unclassified (1) RCC [10] Aquarter of patients with RCC will develop locally advancedor metastatic diseases and a third of patients with localizeddisease at presentation will have recurrence thereafter [11 12]Since themalignant nature and therapeutic response to recentmolecular targeting agents differ among the histological sub-types of RCC it is critical to make a correct diagnosis of renaltumors For example the 5-year survival of RCC is estimatedto be approximately 62 for all stages while that of distantmetastasis decreases to 10 [13] Furthermore a number ofpathological markers have been developed to improve thepoor survival of metastatic RCC [14] Therefore detectionof cytopathological markers simultaneously using multiplefluorescent dyes would be valuable in the pathological diag-nosis to differentiate renal tumors and cancer subtypesWhen a clinician has to make a decision using pathologicalspecimens obtained by needle biopsy for example detectionof several cytopathological markers simultaneously would bevery useful Furthermore it would be an advantage to be ableto reexamine tissue sections again after long-term storageThus the stability of fluorescent dyes is quite important

In order to develop a new technique for immunohis-tochemical staining in the pathological diagnosis of can-cer we examined here tissue sections containing humanrenal tumors by means of quadruple staining using anti-bodies labeled with two Fluolid dyes Fluolid-Green andFluolid-Orange in combination with Alexa Fluor 647 and410158406-diamidino-2-phenylindole (DAPI) Antibodies againstKank1 cytokeratin 7 (CK7) and CD10 proteins were used asthe primary antibodies and Fluolid-conjugated IgG (Kank1and CK7) and Alexa Fluor 647-conjugated IgG (CD10) wereused as the secondary antibodies to detect the primaryantibodies The gene for Kank1 (Kank1) was found to be atumor suppressor gene and its expression was decreased orlost in renal tumors [15] CK7 and CD10 have been used inthe histologic diagnosis of renal tumors [16ndash18] CD10 orneprilysin is a cell-surface glycoprotein expressed in specificsubtypes of renal tumors and has zinc-dependent metallo-protease activity that degrades small secreted peptides suchas the amyloid beta peptide [18] CK7 is a type II keratinexpressed in simple glandular epithelia and in transitionalepithelium CK7 has been used to differentiate chromophobeand papillary RCCs [17]

2 Materials and Methods

21 Reagents A rabbit anti-human cytokeratin 7 (CK7) anti-body was purchased from Funakoshi (Tokyo Japan) a goatanti-human neprilysin (CD10) antibody from RampD Systems(Minneapolis MN) and donkey anti-mouse IgG and donkeyanti-rabbit IgG from Jackson ImmunoResearch (West GrovePA) Alexa Fluor 647-conjugated donkey anti-goat IgG waspurchased from Life Technologies (Carlsbad CA) Fluolid-Orange and Fluolid-Green were purchased from Cosmo Bio(Tokyo Japan)

22 Immunohistochemistry IgG was labeled with each Flu-olid dye using a Fluolid-W proteinantibody labeling kit(International Science Technology Fukuoka Japan) accord-ing to the manufacturerrsquos instructions Briefly IgG wasdissolved into 02M sodium bicarbonate buffer (pH 83)mixedwith a Fluolid dye (dissolved inDMSO) and incubatedfor 2 hr at room temperature Unreacted dye was removedwith a NAP-5 column (GE Healthcare Japan Tokyo Japan)

Paraffin-embedded specimens from RCC patients wereobtained upon written informed consent after approval fromthe ethics committees of Tokyo Medical University andthe National Institute of Advanced Industrial Science andTechnology Specimens were deparaffinized by washing threetimes with fresh xylene for 5min and then washing withgraded ethanol followed by washing twice with phosphate-buffered saline (PBS) For retrieving antigens samples wereboiled in 10mM citrate buffer (pH 60) for 10min using amicrowave oven as reported previously [19] Blocking wasperformed with 5 (vv) donkey serum in 03 Triton-X 100PBS followed by washing twice with PBS An anti-Kank1 monoclonal antibody [20] diluted 1 50 with PBSan anti-CK7 polyclonal antibody diluted 1 100 with PBSand an anti-CD10 polyclonal antibody diluted 1 100 withPBS were used as the primary antibodies The sampleswere incubated with the antibodies overnight at 4∘C andthenwashed three times with PBS Fluolid-Green-conjugatedanti-mouse IgG (1 100) and Fluolid-Orange-conjugated anti-rabbit IgG (1 100) and Alexa Fluor 647-conjugated anti-goatIgG (1 500) were used as the secondary antibodies Thesamples were washed with PBS and stained for 10min with02 120583gmL DAPI (Invitrogen Carlsbad CA) The sampleswere observed using a confocal laser scanning microscope(LSM 510 META Carl Zeiss Oberkochen Germany) underthe following conditions 488 nm excitation500ndash560 nmemission for Fluolid-Green 488 nm580ndash650 nm for Fluolid-Orange and 633 nmLP650 nm for Alexa Fluor 647 Imageswere acquired using LSM510 ver 32 software (Carl Zeiss) andprocessed with Photoshop 70 (Adobe Systems San Jose CA)

3 Results

31 Immunostaining of Renal Tumors with Fluolid-LabeledAntibodies To examine the usefulness of Fluolid dyes inmul-tiple immunostaining we performed immunostaining usingantibodies labeled with two different Fluolid dyes (Fluolid-Green and Fluolid-Orange) Alexa Fluor 647 was used asthe third fluorescent dye and also as a reference Antibodiesagainst Kank1 CK7 or CD10 proteins raised in mice rabbitsor goats respectively were used as the primary antibodiesand fluorescently labeled IgGs for respective species wereused as the secondary antibodies DAPI was included asthe fourth dye to localize nuclei We first performed single-dye immunostaining of kidney tissues to detect Kank1 CK7or CD10 respectively and confirmed that the respectivefluorescence images did not overlap (data not shown) Wethen immunostained normal mouse kidney tissues withmultiple markers (data not shown) and then applied thesystem to immunostain human renal tumors

BioMed Research International 3

Table 1 Summary of immunostaining of Kank1 CK7 and CD10 in renal tumors

Marker Renal tumorClear cell RCC Papillary RCC Chromophobe RCC AML ACD-RCC

Kank1 minus + minus minus +CK7 minus ++ minus minus +CD10 ++ + plusmn + +

The results of simultaneous staining of renal tumors withmultiple markers labeled with Fluolid dyes are shown inFigure 1 In normal kidney tissue Kank1 and CK7 wereexpressed in the cytoplasm of renal tubular cells whileCD10 was expressed in the membrane of renal tubular cells(Figure 1(a)) We applied this quadruple staining system toexamine cancerous areas of clear cell RCC (Figure 1(b)) pap-illary RCC (Figure 1(c)) chromophobe RCC (Figure 1(d))AML (Figure 1(e)) and acquired cystic disease-associatedRCC (ACD-RCC or RCC in dialysis patients) (Figure 1(f))In tumor sections Kank1 was stained in papillary RCC andACD-RCC and weakly or negatively in all other tumors CK7was also positive in papillary RCC and ACD-RCC CD10was expressed in clear cell papillary and ACD-RCCs andAML and weakly in chromophobe RCC These results aresummarized in Table 1

32 Stability of Fluorescence in Pathological Specimens Wealso examined the stability of Fluolid dyes in a sectionthat was stored at room temperature for almost three years(Figure 2) A normal human kidney tissue section observedpreviously (Figure 2(a)) was observed again two years and 11months later (Figure 2(b))The result showed that fluorescentimages of Fluolid dyeswere identical between the original andthe section years later while Alexa Fluor 647 became veryweak

4 Discussion

In this study we examined whether Fluolid dyes are usefulfor pathological diagnosis and showed examples of quadruplestaining of human renal tumors using Fluolid dyes (Fluolid-Green and Fluolid-Orange) along with Alexa Fluor 647 andDAPI Antibodies against Kank1 CK7 and CD10 were usedas the primary antibodies and fluorescently labeled IgGs wereused as the secondary antibodies To effectively separate theimages from different fluorescent dyes it was necessary to setup an appropriate set of fluorescence filtersWefirst examinedthe assay system including the filter set by evaluating whetherthe images were completely separated observing the imagesafter single double or triple staining using all combinationsof Fluolid dyes Alexa Fluor 647 and DAPI (see Section 2)Then we made an assay system for quadruple staining usingall fluorescent dyes

Using this system we performed quadruple staining ofclear cell RCC papillary RCC chromophobe RCC AMLandACD-RCC (Figure 1) Seeing the differences among thesesubtypes of renal tumors is one of the most challenging

differential diagnoses [16] The overlapping nuclear cyto-plasmic and architectural characteristics among these fivetumor subtypes have been well documented by hematoxylinand eosin (HE) staining [16 17] In addition we foundthatmultiple immunohistochemicalmarkerswith differentialexpression in these renal tumors are available (Figures 1(b)ndash1(f))

As summarized inTable 1 wewere able to differentiate theabove subtypes of renal tumors by analysis of the differentialexpression of these markers Here Kank1 was pathologicallyexamined for the first time in RCC subtypes where Kank1was negative in clear cell and chromophobe RCCs and inAML but positive in papillary RCC and ACD-RCC (Table 1)Kank1 was found as a tumor suppressor of clear cell RCC[15] and the locus (9p243) is frequently deleted in clearcell RCC [21] Meanwhile CK7 was positive in papillaryRCC but negative in chromophobe RCC CK7 is localized inthe cytoplasm of most papillary RCC collecting duct RCCand urothelial carcinoma but it is also positive in othertumors [18] While CK7 has been reported to be positive inmany cases of chromophobe RCC 91 for example [17]there were reports of relatively low frequencies such as66 [22] suggesting the contribution of genetic variationsamong patients with chromophobe RCC Notably there is apossibility that its diffuse andmembranous stainingmayneedfurther improvement in fluorescent detection especially inenhancing the brightness of images AML is thought to be abenign tumor and thus surgical treatment is not necessary ifthe tumor is correctively diagnosed by computed tomography(CT) or ultrasonography However it is sometimes difficultto differentiate it from malignant tumors when the tumoris small or there is no fatty component [23] Therefore itmay be helpful to distinguish AML from other malignanttumors in a small specimen In our study CK7 was negativein AML as reported previously [24] It was reported that CK7is negative in ACD-RCC [25] although it was positive in ourstudy (Table 1)

CD10 is a surface glycoprotein identified in a varietyof healthy cells where it hydrolyzes peptide bonds anddecreases the cellular response to local peptide hormones Inthe normal kidney CD10 is strongly expressed at proximaltubular cell brush borders [18] CD10 is strongly and diffuselyexpressed in renal cell neoplasms derived from proximaltubules in clear cell RCC and may be present in papillaryRCC usually not in a diffusemanner but in a luminal patternCD10 is also expressed in vascular cells of AML [26] andin ACD-RCC [27] but is usually negative in chromophobeRCC [28] Note that CK7 and CD10 are sometimes used incombination to differentiate subtypes of RCC [16]


DAPI Merged image HE120572Kank1Fluolid-Green

120572CK7Fluolid-Orange

120572CD10AlexaFluor 647

(a)




(b)




(c)




(d)




(e)




(f)

Figure 1 Immunostaining of renal tumors Antibodies against Kank1 (120572Kank1) CK7 (120572CK7) and CD10 (120572CD10) were used as the primaryantibodies and Fluolid-Green-conjugated donkey anti-mouse IgG Fluolid-Orange-conjugated donkey anti-rabbit IgG and Alexa Fluor647-conjugated donkey anti-goat IgG were used respectively as the secondary antibodies DAPI was used to stain nuclei and tissues werehistopathologically examined by hematoxylin and eosin (HE) staining Results of immunostaining of normal tissue (a) clear cell RCC (b)papillary RCC (c) chromophobe RCC (d) renal angiomyolipoma (e) and acquired cystic disease-associated RCC (f) are shown Note thatthe parts shown by HE staining in cancer do not match with those shown by immunostaining Image magnification times40 for immunostainingin (a)ndash(f) and HE staining in (a) times20 for HE staining in (b)ndash(f) Bar = 20120583m


DAPI Merged image120572Kank1Fluolid-Green



(a)




(b)

Figure 2 Stability of fluorescent dyes in histopathological sections Fluorescent images were examined for a tissue section before (a) orafter (b) the storage at room temperature for almost three years A normal human kidney tissue section was subjected to quadruple stainingwith three different Fluolid dyes and DAPI Antibodies against Kank1 CK7 and CD10 were used as the primary antibodies and Fluolid-Green-conjugated anti-mouse IgG Fluolid-Orange-conjugated anti-rabbit IgG and Alexa Fluor 647-conjugated anti-goat IgG were used asthe secondary antibodies Bar = 20120583m

We also examined the stability of fluorescently labeledmarkers on a section that was stored at room temperaturefor almost three years (Figure 2) The fluorescent imagesof Fluolid dyes were identical between the original and thesection years later while that of Alexa Fluor 647 becameweakTherefore Fluolid dyes are advantageous for long-termpreservation of stained histopathological sections

Multiple immunoenzyme staining systems have beendeveloped by modifying standard chromogen-based immu-noenzyme techniques Double staining systems were devel-oped to achieve maximum color contrast between red (suchas 3-amino-9-ethyl-carbazole) and brown (such as 331015840-diaminobenzidine or DAB) by unmixing spectral images[29] However colorimetric separation of signals amongcolocalizing markers and between markers and endogenouspigments is almost impossible and so only double stainingor triple staining for limited cases [29] can be applied Incontrast a variety of images of different markers can beseparated with good contrast (high signalnoise ratios) andsensitivity (low background signals) in immunofluorescenttechniques However the storage of stained specimens ismostly limited to daysweeks due to poor stability of fluores-cent dyes Here we provided a double staining system withstable fluorescent dyes However this is only a progress in theprocess to develop a future immunostaining systemwhere thethird stable fluorescent dye with a complete set of markersand automated less expensive devices are available A newFluolid dye compatible with Alexa Fluor 647 is now underdevelopment to provide an immunohistochemical systemwith three stable markers

In this study we provided examples of quadruple stainingof sections of human renal tumors using Fluolid dyes AlexaFluor 647 and DAPI In conclusion immunostaining with

multiple fluorescently labeled markers will improve the clas-sification of renal tumor subtypes using limited amounts ofsamples whichmay be of particular help for pathologists andclinicians Furthermore Fluolid dyes will enable long-termpreservation of stained histopathological sections Hopefullythis multiple staining method will improve the accuracy ofthe diagnosis of various diseases

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

Dilibaier Wuxiuer and Yun Zhu contributed equally to thispaper

Acknowledgments

This research was supported partly by a Special CoordinationFund for Promoting Science and Technology (EncouragingDevelopment of Strategic Research Centers) and a Knowl-edge Cluster Initiative program and a Grant-in-aid for BasicAreas from the Ministry of Education Culture SportsScience and Technology of Japan

References

[1] D W Hedley M Pintilie J Woo et al ldquoUp-regulation of theredox mediators thioredoxin and apurinicapyrimidinic exci-sion (APE)Ref-1 in hypoxic microregions of invasive cervicalcarcinomas mapped using multispectral wide-field fluores-cence image analysisrdquo The American Journal of Pathology vol164 no 2 pp 557ndash565 2004


[2] MBalicH Lin L Young et al ldquoMost early disseminated cancercells detected in bone marrow of breast cancer patients havea putative breast cancer stem cell phenotyperdquo Clinical CancerResearch vol 12 no 19 pp 5615ndash5621 2006

[3] E L Snyder D Bailey M Shipitsin K Polyak and MLoda ldquoIdentification of CD44v6(+)CD24- breast carcinomacells in primary human tumors by quantum dot-conjugatedantibodiesrdquo Laboratory Investigation vol 89 no 8 pp 857ndash8662009

[4] M Kai H Onishi M Souzaki et al ldquoSemi-quantitative eval-uation of CD44(+)CD24(-) tumor cell distribution in breastcancer tissue using a newly developed fluorescence immuno-histochemical staining methodrdquo Cancer Science vol 102 no 12pp 2132ndash2138 2011

[5] G Li M Guillaud M Follen and C MacAulay ldquoDoublestaining cytologic samples with quantitative feulgen-thioninand Anti-Ki-67 immunocytochemistry as a method of distin-guishing cells with abnormalDNA content fromnormal cyclingcellsrdquo Analytical and Quantitative Cytology and Histology vol34 no 5 pp 273ndash284 2012

[6] R Kiyama Y Zhu and T Aoyagi ldquoGenetics of renal tumorsrdquoin Renal Tumors J Chen Ed pp 3ndash30 InTech Rijeka Croatia2013

[7] Y Zhu T Ogaeri J-I Suzuki et al ldquoApplication of fluolid-orange-labeled probes for DNAmicroarray and immunologicalassaysrdquo Biotechnology Letters vol 33 no 9 pp 1759ndash1766 2011

[8] T Kanemaru K Hirata S-I Takasu et al ldquoA fluorescencescanning electron microscoperdquo Ultramicroscopy vol 109 no 4pp 344ndash349 2009

[9] A Matsuda T Matsuda Japan Cancer Surveillance ResearchGroup et al ldquoCancer incidence and incidence rates in Japanin 2007 a study of 21 population-based cancer registries forthe monitoring of cancer incidence in Japan (MCIJ) projectrdquoJapanese Journal of Clinical Oncology vol 43 no 3 pp 328ndash3362013

[10] I Rosner G Bratslavsky P A Pinto and W M Linehan ldquoTheclinical implications of the genetics of renal cell carcinomardquoUrologic Oncology vol 27 no 2 pp 131ndash136 2009

[11] H T Cohen and F J McGovern ldquoRenal-cell carcinomardquo TheNew England Journal of Medicine vol 353 no 23 pp 2477ndash2490 2005

[12] B R Lane and M W Kattan ldquoPrognostic models and algo-rithms in renal cell carcinomardquo Urologic Clinics of NorthAmerica vol 35 no 4 pp 613ndash625 2008

[13] C S Ng C GWood PM Silverman NM Tannir P Tamboliand C M Sandler ldquoRenal cell carcinoma diagnosis stagingand surveillancerdquo The American Journal of Roentgenology vol191 no 4 pp 1220ndash1232 2008

[14] G D Stewart F C OrsquoMahony T Powles A C Riddick DJ Harrison and D Faratian ldquoWhat can molecular pathologycontribute to the management of renal cell carcinomardquo NatureReviews Urology vol 8 no 5 pp 255ndash265 2011

[15] N Kakinuma Y Zhu Y Wang B C Roy and R KiyamaldquoKank proteins structure functions and diseasesrdquo Cellular andMolecular Life Sciences vol 66 no 16 pp 2651ndash2659 2009

[16] S K Tickoo and A Gopalan ldquoPathologic features of renalcortical tumorsrdquo Urologic Clinics of North America vol 35 no4 pp 551ndash561 2008

[17] H A Al-Ahmadie D Alden SW Fine et al ldquoRole of immuno-histochemistry in the evaluation of needle core biopsies in adultrenal cortical tumors an ex vivo studyrdquo The American Journalof Surgical Pathology vol 35 no 7 pp 949ndash961 2011

[18] L D Truong and S S Shen ldquoImmunohistochemical diagnosisof renal neoplasmsrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 1 pp 92ndash109 2011

[19] S R Shi M E Key and K L Kalra ldquoAntigen retrievalin formalin-fixed paraffin-embedded tissues an enhance-ment method for immunohistochemical staining based onmicrowave oven heating of tissue sectionsrdquo Journal of Histo-chemistry and Cytochemistry vol 39 no 6 pp 741ndash748 1991

[20] B C Roy T Aoyagi S Sarkar et al ldquoPathological charac-terization of Kank in renal cell carcinomardquo Experimental andMolecular Pathology vol 78 no 1 pp 41ndash48 2005

[21] Y Sato T Yoshizato Y Shiraishi et al ldquoIntegrated molecularanalysis of clear-cell renal cell carcinomardquo Nature Genetics vol45 no 8 pp 860ndash867 2013

[22] B P Adley V Papavero J Sugimura B T Teh and X JYang ldquoDiagnostic value of cytokeratin 7 and parvalbuminin differentiating chromophobe renal cell carcinoma fromrenal oncocytomardquo Analytical and Quantitative Cytology andHistology vol 28 no 4 pp 228ndash236 2006

[23] J K Kim S Y Park JH Shon andK S Cho ldquoAngiomyolipomawith minimal fat differentiation from renal cell carcinoma atbiphasic helical CTrdquo Radiology vol 230 no 3 pp 677ndash6842004

[24] Q Xu Q Cao N Liu Z Fang Z Ye and T Peng ldquoRenalcollecting duct carcinoma with extensive coagulative necrosismimicking anemic infarct report of a case and the literaturereviewrdquo Diagnostic Pathology vol 8 no 1 article 119 2013

[25] N Kuroda A Tanaka C Ohe and Y Nagashima ldquoRecentadvances of immunohistochemistry for diagnosis of renaltumorsrdquo Pathology International vol 63 no 8 pp 381ndash3902013

[26] S E Hohensee F G La Rosa P Homer et al ldquoRenal epithe-lioid angiomyolipoma with a negative premelanosome markerimmunoprofile a case report and review of the literaturerdquoJournal of Medical Case Reports vol 7 no 1 article 118 2013

[27] S Ahn G Y Kwon Y M Cho et al ldquoAcquired cystic disease-associated renal cell carcinoma further characterization ofthe morphologic and immunopathologic featuresrdquo MedicalMolecular Morphology vol 46 no 4 pp 225ndash232 2013

[28] S Yasir L Herrera C Gomez-Fernandez et al ldquoCD10(+) andCK7RON(-) immunophenotype distinguishes renal cell carci-noma conventional type with eosinophilic morphology fromits mimickersrdquo Applied Immunohistochemistry and MolecularMorphology vol 20 no 5 pp 454ndash461 2012

[29] C M van der Loos ldquoMultiple immunoenzyme stainingmethods and visualizations for the observation with spectralimagingrdquo Journal of Histochemistry and Cytochemistry vol 56no 4 pp 313ndash328 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Renal cell carcinoma (RCC) is the most common type ofkidney cancer and is classified into five histologic subtypesclear cell (70ndash80) papillary (10ndash15) chromophobe (3ndash5) collecting duct (1) and unclassified (1) RCC [10] Aquarter of patients with RCC will develop locally advancedor metastatic diseases and a third of patients with localizeddisease at presentation will have recurrence thereafter [11 12]Since themalignant nature and therapeutic response to recentmolecular targeting agents differ among the histological sub-types of RCC it is critical to make a correct diagnosis of renaltumors For example the 5-year survival of RCC is estimatedto be approximately 62 for all stages while that of distantmetastasis decreases to 10 [13] Furthermore a number ofpathological markers have been developed to improve thepoor survival of metastatic RCC [14] Therefore detectionof cytopathological markers simultaneously using multiplefluorescent dyes would be valuable in the pathological diag-nosis to differentiate renal tumors and cancer subtypesWhen a clinician has to make a decision using pathologicalspecimens obtained by needle biopsy for example detectionof several cytopathological markers simultaneously would bevery useful Furthermore it would be an advantage to be ableto reexamine tissue sections again after long-term storageThus the stability of fluorescent dyes is quite important

In order to develop a new technique for immunohis-tochemical staining in the pathological diagnosis of can-cer we examined here tissue sections containing humanrenal tumors by means of quadruple staining using anti-bodies labeled with two Fluolid dyes Fluolid-Green andFluolid-Orange in combination with Alexa Fluor 647 and410158406-diamidino-2-phenylindole (DAPI) Antibodies againstKank1 cytokeratin 7 (CK7) and CD10 proteins were used asthe primary antibodies and Fluolid-conjugated IgG (Kank1and CK7) and Alexa Fluor 647-conjugated IgG (CD10) wereused as the secondary antibodies to detect the primaryantibodies The gene for Kank1 (Kank1) was found to be atumor suppressor gene and its expression was decreased orlost in renal tumors [15] CK7 and CD10 have been used inthe histologic diagnosis of renal tumors [16ndash18] CD10 orneprilysin is a cell-surface glycoprotein expressed in specificsubtypes of renal tumors and has zinc-dependent metallo-protease activity that degrades small secreted peptides suchas the amyloid beta peptide [18] CK7 is a type II keratinexpressed in simple glandular epithelia and in transitionalepithelium CK7 has been used to differentiate chromophobeand papillary RCCs [17]

2 Materials and Methods

21 Reagents A rabbit anti-human cytokeratin 7 (CK7) anti-body was purchased from Funakoshi (Tokyo Japan) a goatanti-human neprilysin (CD10) antibody from RampD Systems(Minneapolis MN) and donkey anti-mouse IgG and donkeyanti-rabbit IgG from Jackson ImmunoResearch (West GrovePA) Alexa Fluor 647-conjugated donkey anti-goat IgG waspurchased from Life Technologies (Carlsbad CA) Fluolid-Orange and Fluolid-Green were purchased from Cosmo Bio(Tokyo Japan)

22 Immunohistochemistry IgG was labeled with each Flu-olid dye using a Fluolid-W proteinantibody labeling kit(International Science Technology Fukuoka Japan) accord-ing to the manufacturerrsquos instructions Briefly IgG wasdissolved into 02M sodium bicarbonate buffer (pH 83)mixedwith a Fluolid dye (dissolved inDMSO) and incubatedfor 2 hr at room temperature Unreacted dye was removedwith a NAP-5 column (GE Healthcare Japan Tokyo Japan)

Paraffin-embedded specimens from RCC patients wereobtained upon written informed consent after approval fromthe ethics committees of Tokyo Medical University andthe National Institute of Advanced Industrial Science andTechnology Specimens were deparaffinized by washing threetimes with fresh xylene for 5min and then washing withgraded ethanol followed by washing twice with phosphate-buffered saline (PBS) For retrieving antigens samples wereboiled in 10mM citrate buffer (pH 60) for 10min using amicrowave oven as reported previously [19] Blocking wasperformed with 5 (vv) donkey serum in 03 Triton-X 100PBS followed by washing twice with PBS An anti-Kank1 monoclonal antibody [20] diluted 1 50 with PBSan anti-CK7 polyclonal antibody diluted 1 100 with PBSand an anti-CD10 polyclonal antibody diluted 1 100 withPBS were used as the primary antibodies The sampleswere incubated with the antibodies overnight at 4∘C andthenwashed three times with PBS Fluolid-Green-conjugatedanti-mouse IgG (1 100) and Fluolid-Orange-conjugated anti-rabbit IgG (1 100) and Alexa Fluor 647-conjugated anti-goatIgG (1 500) were used as the secondary antibodies Thesamples were washed with PBS and stained for 10min with02 120583gmL DAPI (Invitrogen Carlsbad CA) The sampleswere observed using a confocal laser scanning microscope(LSM 510 META Carl Zeiss Oberkochen Germany) underthe following conditions 488 nm excitation500ndash560 nmemission for Fluolid-Green 488 nm580ndash650 nm for Fluolid-Orange and 633 nmLP650 nm for Alexa Fluor 647 Imageswere acquired using LSM510 ver 32 software (Carl Zeiss) andprocessed with Photoshop 70 (Adobe Systems San Jose CA)

3 Results

31 Immunostaining of Renal Tumors with Fluolid-LabeledAntibodies To examine the usefulness of Fluolid dyes inmul-tiple immunostaining we performed immunostaining usingantibodies labeled with two different Fluolid dyes (Fluolid-Green and Fluolid-Orange) Alexa Fluor 647 was used asthe third fluorescent dye and also as a reference Antibodiesagainst Kank1 CK7 or CD10 proteins raised in mice rabbitsor goats respectively were used as the primary antibodiesand fluorescently labeled IgGs for respective species wereused as the secondary antibodies DAPI was included asthe fourth dye to localize nuclei We first performed single-dye immunostaining of kidney tissues to detect Kank1 CK7or CD10 respectively and confirmed that the respectivefluorescence images did not overlap (data not shown) Wethen immunostained normal mouse kidney tissues withmultiple markers (data not shown) and then applied thesystem to immunostain human renal tumors







4 Discussion










(a)




(b)




(c)




(d)




(e)




(f)






(a)




(b)










Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Discussion










(a)




(b)




(c)




(d)




(e)




(f)






(a)




(b)










Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















(a)




(b)




(c)




(d)




(e)




(f)






(a)




(b)










Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















(a)




(b)










Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article development of pathological diagnostics...

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