evaluation of decalcification techniques for rat femurs...

Research ArticleEvaluation of Decalcification Techniques for Rat Femurs UsingHE and Immunohistochemical Staining

Haixia Liu1 Ruyuan Zhu1 Chenyue Liu2 Rufeng Ma1 Lili Wang1

Beibei Chen1 Lin Li1 Jianzhao Niu1 Dandan Zhao3 Fangfang Mo3

Min Fu4 Dieter Broumlmme5 Dongwei Zhang3 and Sihua Gao3

1Preclinical Medicine School Beijing University of Chinese Medicine Beijing 100029 China2Chinese Material Medica School Beijing University of Chinese Medicine Beijing 100029 China3Diabetes Research Center Beijing University of Chinese Medicine Beijing 100029 China4The Research Institute of McGill University Health Center Montreal QC Canada H4A 3J15Oral Biological Medicinal Science University of British Columbia Vancouver BC Canada V6T 1Z3

Correspondence should be addressed to Dongwei Zhang dongwei1006gmailcom and Sihua Gao gaosihua1216163com

Received 16 November 2016 Accepted 20 December 2016 Published 26 January 2017

Academic Editor Alexander N Orekhov

Copyright copy 2017 Haixia Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aim In routine histopathology decalcification is an essential step for mineralized tissues The purpose of this study is to evaluatethe effects of different decalcification solutions on the morphological and antigenicity preservation in Sprague Dawley (SD) ratfemursMaterials and Methods Four different decalcification solutions were employed to remove the mineral substances from ratfemurs including 10 neutral buffered EDTA 3 nitric acid 5 nitric acid and 8 hydrochloric acidformic acid Shaking andlow temperature were used to process the samplesThe stainings of hematoxylin-eosin (HE) and immunohistochemical (IHC) wereemployed to evaluate the bone morphology and antigenicity Key Findings Different decalcification solutions may affect the qualityof morphology and the staining of paraffin-embedded sections in pathological examinations Among four decalcifying solutions3 nitric acid is the best decalcifying agent for HE staining 10 neutral buffered EDTA and 5 nitric acid are the preferreddecalcifying agents for IHC staining Significance The current study investigated the effects of different decalcifying agents on thepreservation of the bone structure and antigenicity which will help to develop suitable protocols for the analyses of the bony tissue

1 Introduction

Decalcification is an essential step routinely performed forhistopathological observation of bone and bone-containingtissues [1] Problems arise during tissue sectioning and pro-cessing because of the mineral content in a densely packedorganic extracellular matrix structure consisting of both col-lagenous and noncollagenous materials [2] Minerals mainlyin form of calcium and phosphorus insoluble salts calledhydroxyapatite (HA) account for sixty-five percent of bonetissue [3 4] HA crystals bound to the organic protein matrixprovide the bone hardness and are the cause of the resistanceduring tissue cutting using regular microtomes [5] In addi-tion the current decalcifying methods are characterized bylaborious procedures and the frequent loss of immunoreac-tivity [1] which may impede genuine understanding of bone

remodeling in the development of skeletal diseases There-fore the development of an efficient decalcifying method isan ongoing challenge for a high-quality processing of paraf-finized bone samples

Efficient decalcification protocols will allow the removalof insolvable inorganic salts from bony tissues which willsoften bone and teeth for easy sectioning [6] Currently thereare several decalcification solutions available which includeinorganic and organic acids a neutral fluid containing achelating agent or a mixture of solutions [7ndash9] An idealdecalcifying approach is to preserve the tissue morphologyand antigenicity [9] Low processing temperature low acidityof the decalcification solution and continuous sample shak-ing contribute to an efficient decalcifying and preserving thetissue structure and antigenicity of the samples [10ndash12]

HindawiBioMed Research InternationalVolume 2017 Article ID 9050754 6 pageshttpsdoiorg10115520179050754

2 BioMed Research International

Hematoxylin-eosin (HE) staining is one of the principalstains in histopathology and the most widely used stain inmedical diagnosis Immunohistochemistry refers to the pro-cess of detecting antigens (eg proteins) in cells of a tissuesection by exploiting the principle of antibodies bindingspecifically to antigens in biological tissues to realize thequalitative and quantitative analysis of antigen [13] Insulin-like growth factor 1 (IGF-1) the most abundant growth factorin bone has the endocrine and paracrine actions during theprocess of bone remodeling [14] In addition IGF-1 positivelycorrelates with bone mineral density in osteoporosis and isimportant for the investigation of bone formation [15]

In order to find the most suitable decalcifying agent weevaluated four different decalcifying solutions for the wholefemurs of adult rats by evaluating the time of decalcifica-tion ease of tissue slicing morphological preservation withHE staining and the preservation of antigenicity for boneproteins such as IGF-1 using immunohistochemical (IHC)staining

2 Materials and Methods

21 Chemicals and Antibody All chemical reagents werepurchased from Beijing Sinopharm Chemical Reagents CoLtd (Beijing China) Rabbit polyclonal IGF-1 antibody wasfrom Santa Cruz Biotechnology Inc (Cat sc-9013 TexasUSA)

22 Animal Feeding Six 16-week-old female Sprague Daw-ley (SD) rats (300 plusmn 10 g) were purchased from ChinaHuafukang Animal Technology Co Ltd (License numberSCXK (Beijing) 2011-0004 Beijing China) Animals werehoused at clean level conditions (certification number SCXK(Jing) 2011-0024) at Beijing University of Chinese Medicine(BUCM) with the temperature of 22 plusmn 1∘C humidity of 55 plusmn5 and a 12 h lightdark cycle All rats were allowed freeaccess to tapwater and foodThe study protocol was approvedby the animal care committee of BUCM China

After one week of acclimation the rats were anes-thetized with pentobarbital sodium (1 sodium pentobarbi-tal 04mL100 g ip) The bilateral femurs were harvestedand stored at minus80∘C for further analyses

23 Decalcification The femurs were prefixed in 4 para-formaldehyde for 24 h Samples were then rinsed in runningtap water for 24 h and incubated with four different decalci-fying solutions (Table 1) (1) 10 EDTA (pH 74) (2) 3 nitricacid (3) 5 nitric acid and (4) 8 hydrochloric acidformicacid followed by neutralizing with 01 aqueous ammoniasolution for 30min Decalcification was performed at 4∘Cunder continuous shaking The decalcifying solutions werechanged on a daily basis and the total decalcification timewasrecorded

The decalcification process was ended when the bonewas easily penetrated through by a needle without any forceSubsequently samples were washed in running tap water for24 h and then followed by routine dehydration and paraffinembedding 5 120583m sections were cut using a Leica microtome

(Leica Germany) and placed on adhesive-precoated (5poly-L-lysine) glass slides

The quality of decalcification was evaluated by the fol-lowing criteria (1) the time of decalcification (2) the ease ofsectioning (3) the morphological preservation by HE stain-ing (4) the antigenicity preservation by IHC staining Theease of sectioning and morphological preservation weregraded from 1 to 4 (1 poor 2 fair 3 good and 4 excellent)[16]

24 HE Staining HE staining was conducted according toroutine protocols [17] Briefly after deparaffinization andrehydration 5120583m longitudinal sections were stained withhematoxylin solution for 5min followed by 5 dips in 1 acidethanol (1 HCl in 70 ethanol) and then rinsed in distilledwater Then the sections were stained with eosin solution for3min and followed by dehydration with graded alcohol andclearing in xylene The mounted slides were then examinedand photographed using an Olympus BX53 fluorescencemicroscope (Tokyo Japan) The staining intensity of thetrabecular bone was analyzed by Image-Pro Plus 60 softwareand expressed as IOD value

25 Immunohistochemical Staining 5 120583m longitudinal sec-tions of the paraffin-embedded femurs were kept at 60∘Cfor 24 h in the oven and then followed by deparaffinizingwith xylene and hydrating with an ethanol gradient (100ndash70) After successively incubating with antigen retrievalsolution (Shanghai Shunbai BiotechnologyCompany Shang-hai China) and 3 H

2O2for 30min the slides were rinsed

with water and incubated with the primary antibody (IGF-1(1 50)) overnight at 4∘C For negative controls the primaryantibody was replaced by nonimmunized serum The nextday the slides were rinsed and incubated with the corre-sponding secondary antibody (Beijing Biosynthesis Biotech-nology Co Ltd Beijing China) for 30min followed by 331015840-diaminobenzidine (DAB) and hematoxylin staining respec-tively The slides were then examined and photographedusing an Olympus BX53 fluorescence microscope (TokyoJapan)TheDAB stainingwas analyzed by Image-Pro Plus 60software

26 Statistics Analysis One-way ANOVA was used to evalu-ate the effect of decalcifying solutions in the quantitative anal-yses of HE staining and IGF-1 antigenicity preservation Sixsections from different rats were taken for histological analy-ses Studentrsquos 119905-test was used to analyze the difference betweengroups A value of 119901 lt 005 was considered significant and119901 lt 001 considered statistically significant

3 Results

31 Time of Decalcification Decalcification in 10 EDTA(pH 74) required 21 days The other decalcifying solutionsneeded 8 days for decalcification with no obvious differencesin the decalcification time

BioMed Research International 3

Table 1 The ingredients and preparation of different decalcifying agents

Decalcifying solution 1 Decalcifying solution 2 Decalcifying solution 3 Decalcifying solution 4Decalcifyingagents 10 EDTA 3 nitric acid 5 nitric acid 8 hydrochloric

acidformic acid

Preparation 100 g EDTA and 10 gsodium hydroxide 3mL nitric acid 5mL nitric acid 8mL hydrochloric acid and

8mL formic acidDistilledwater Add to 1000mL Add to 100mL Add to 100mL Add to 100mL

pH 74 mdash mdash mdash

12

3

(a)

10 EDTA 8 HFA

100x

200x

3HNO3 5HNO3

200 120583m 200 120583m 200 120583m 200 120583m

100 120583m100 120583m100 120583m100 120583m

(b)

Figure 1 Representative microstructure ((a) times100) and histological images (b) of HE staining showed the influences of different decalcifiedsolutions on morphological structure preservation of rat femurs 1 to 3 represent superficial cartilage and subchondral zone respectivelyHFA represents hydrochloric acidformic acid

32 Ease of Sectioning Themicrostructure of rat femurs (HEstaining) is shown in Figure 1(a) including superficial zonecalcified cartilage zone and subchondral bone The sectionswill be easily cut if bony tissues are well decalcified Thequality of morphological preservation in the section is evalu-ated from trabecular bone staining cartilage tissue stainingand contrast ratio As shown in Table 2 and Figure 1(b)different decalcification solutions resulted in differences inthe easiness of tissue cutting andmorphological preservationThe following order for cutting easiness was observed 8hydrochloric acidformic acid = 3 nitric acid gt 5 nitricacid = 10 EDTA (pH 74) In terms of the quality ofmorphological structure preservation 3 nitric acid was thebest followed by 5 nitric acid and then 10 EDTA (pH 74)and 8 hydrochloric acidformic acid subsequently

33 Morphological Preservation As shown in Figures 1(b)2(a) and 2(b) HE staining demonstrated that differentdecalcifying solutions affected the staining intensity of thesections The methods including 10 EDTA (pH 74) 5nitric acid and 8 hydrochloric acidformic acid had verysimilar effects on the staining intensity of slides However3 nitric acid resulted in the best brightness and uniformityof staining And the sections processed by 8 hydrochloricacidformic acid showed uneven staining

34 Antigenicity Preservation The preservation of antigenwas evaluated by IHC staining As shown in Figure 3 thepositive signals (brown particles) were best observed after

decalcifying with 5 nitric acid and 10 EDTA (pH 74) Theresults indicate that the decalcification solution of 10 EDTA(pH 74) and 5 nitric acid had a better capacity in retainingantigen preservation of IGF-1 in comparison with that of 3nitric acid and 8 hydrochloric acidformic acid

4 Discussion

In the present study we compared the outcome of fourdifferent decalcifying solutions using SD rat femurs on thequality of HE and IHC staining With regard to the HEstaining and morphology preservation 3 nitric acid givesbetter results than 10 EDTA (pH 74) 5 nitric acid and8 hydrochloric acidformic acid In the case of preservingthe antigenicity of the tissue samples 10 EDTA (pH 74) isfound to be the most optimal solution followed by 5 nitricacid 3 nitric acid and 8 hydrochloric acidformic acid

EDTA is one of the most commonly used decalcifyingagents It preserves well antigenicity as shown in this studyHowever the advantage of EDTA as a decalcifying agent ina routine setting is masked by time-consuming incubationespecially for large-sized samples [1 18] Decalcification timereported for EDTA ranges from 2 to 4 months [17] Inaddition most of the investigators used EDTA for decalcifi-cation at room temperature which may damage the antigenavailability of the samples In the current study two variationsof the standard protocols were utilized to improve bonedecalcification quality (1) continuous shaking of the samplesand (2) decalcification at 4∘C The improvements appeared


Table 2 Decalcifying solutions scores as the measurement of ease of sectioning and morphological preservation

Decalcifying solutions Ease of sectioning HE morphological evaluation Total scoreTBS CTS CT Total

10 EDTA 3 3 3 3 9 123 nitric acid 4 4 3 4 12 165 nitric acid 3 3 4 3 10 138 HFA 4 2 4 3 7 12Note TBS trabecular bone staining CTS cartilage tissue staining CT contrast ratio HFA hydrochloric acidformic acid

10 EDTA 8 HFA

200x

400x

3HNO3 5HNO3

100 120583m100 120583m100 120583m100 120583m

50 120583m 50 120583m 50 120583m 50 120583m

(a)

020

015

010

005

0HE

stai

ning

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3

lowast

(b)

Figure 2 Representative images (a) and histological images analysis (b) ofHE staining showed the influences of different decalcified solutionson staining quality of rat femurs Image-Pro Plus was used to quantify the relative IOD value of HE staining of the trabecular bone lowast119901 lt 005compared with EDTA group

400x

400x

10 EDTA

8 HFA

3HNO3

5HNO3

50120583m 50 120583m

50 120583m50 120583m

(a)

03

02

01

0

IGF-

1 ex

pres

sion

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3

lowastlowastlowastlowast

(b)

Figure 3 Representative immunohistochemical images (a) and histological images analysis (b) of IGF-1 expression in rat femurs showed theinfluences of different decalcified solutions on antigenicity preservation Image-Pro Plus was used to analyze the relative IOD value of IGF-1lowastlowast119901 lt 001 compared with EDTA group

to allow for a better and even infiltration of the decalcifyingsolutions into the tissue

An effective decalcifying method permits the generationof morphologically high-quality tissue sections In the cur-rent study we found that 8 hydrochloric acidformic acidand 3 nitric acid are optimal to obtain 5 120583m sections ofgood quality On the other hand 3 nitric acid providesthe best results regarding tissue structure preservation andcontrast ratio Ying et al [19] found that addition of ethanol

to decalcifying agent contributed to a further improvementof the HE staining quality We also found that the addition ofethanol to mixed acids (6mL nitric acid 10mL hydrochloricacid 30mL formic acid 5mL glacial acetic acid 104mL 70ethanol and 45mL water) improved the brightness of thesections with HE staining when compared to that of 10EDTA (data not shown) However the addition of ethanolmay damage antigenicity because no positive signal wasobserved during IHC staining


The effect of different decalcifying agents on antigenpreservation is obviously different In our study decalcifyingwith 5 nitric acid and 10 EDTA (pH 74) produced strongpositive signals of IGF-1 staining In addition Athanasou etal demonstrated that prolonged decalcification in strong acidmay diminish the antigen activity but the weaker acids maynevertheless better preserve the antigenic reactivity mor-phology and staining quality allowing for time consumingfor decalcifying [20]However prolonged decalcificationmayalso adversely affect the staining quality [21] Therefore anaccurate control of the decalcification duration improves thequality of sectioning and staining

Sangeetha et al reported that decalcification in 5 nitricacid resulted in yellow discoloration of the bone sections atambient temperature [9] Furthermoremicrowave techniquemay accelerate the decalcification [9] but the sections arevulnerable to disintegration and falling off from the slides

In order to improve the quality of staining [8] we alsoneutralized the samples after decalcification with mineralacids Contrary to the expectation the sections that weredecalcified in 3 or 5 nitric acid were easily to fall off fromthe slides after neutralization Moreover there was no differ-ence in respect to the quality of the staining and antigenicitypreservation

In summary different decalcification solutionsmay affectthe quality of the morphology and staining of paraffin-embedded bone tissue sections Among the four methodsused in this study 3 nitric acid is the best decalcifyingsolution for HE staining while 10 neutral buffered EDTAand 5 nitric acid are the best decalcifying agent for IHCstaining

Disclosure

The funding sources have no role in study design dataanalysis drafting and submitting the article

Competing Interests

The authors declare no conflict of interests regarding thepublication of this paper

Authorsrsquo Contributions

Haixia Liu and Ruyuan Zhu equally contributed

Acknowledgments

This work was supported by grants from National NaturalScience Foundation ofChina (NSFC81274041NSFC81273995)international cooperation projects of MOE (2011DFA30920)and key drug development programofMOST (20122X09103201-005) as well as the 111 project of MOE (B07007)

References

[1] J C Alers P-J Krijtenburg K J Vissers and H Van DekkenldquoEffect of bone decalcification procedures on DNA in situ

hybridization and comparative genomic hybridization EDTAis highly preferable to a routinely used acid decalcifierrdquo Journalof Histochemistry and Cytochemistry vol 47 no 5 pp 703ndash7101999

[2] W A M E Schrijver P van der Groep L D Hoefnagel et alldquoInfluence of decalcification procedures on immunohistochem-istry andmolecular pathology in breast cancerrdquoModern Pathol-ogy vol 29 pp 1460ndash1470 2016

[3] MDermience G Lognay FMathieu and P Goyens ldquoEffects ofthirty elements on bone metabolismrdquo Journal of Trace Elementsin Medicine and Biology vol 32 pp 86ndash106 2015

[4] L Ciocca I G Lesci O Mezini et al ldquoCustomized hybrid bio-mimetic hydroxyapatite scaffold for bone tissue regenerationrdquoJournal of Biomedical Materials Research Part B Applied Bioma-terials 2015

[5] M D Rohrer and C C Schubert ldquoThe cutting-grinding techni-que for histologic preparation of undecalcified bone and bone-anchored implants Improvements in instrumentation and pro-ceduresrdquo Oral Surgery Oral Medicine Oral Pathology vol 74no 1 pp 73ndash78 1992

[6] W H Lan H W Kwan and I Sunada ldquoSlicing technique fortooth specimens in histological preparationrdquo The Bulletin ofTokyo Medical and Dental University vol 33 no 4 pp 129ndash1361986

[7] H Hatta K Tsuneyama K Nomoto et al ldquoA simple and rapiddecalcification procedure of skeletal tissues for pathology usingan ultrasonic cleaner with D-mannitol and formic acidrdquo ActaHistochemica vol 116 no 5 pp 753ndash757 2014

[8] A Rabiei E Esfandiary M Hajian et al ldquoPlastination of decal-cified bone by a new resin techniquerdquo Advanced BiomedicalResearch vol 3 article no 18 2014

[9] R Sangeetha K Uma and V Chandavarkar ldquoComparison ofroutine decalcificationmethods withmicrowave decalcificationof bone and teethrdquo Journal of Oral and Maxillofacial Pathologyvol 17 no 3 pp 386ndash391 2013

[10] Z Xiong H Ding C Li Y Tian and Z Xu ldquoEffect of decalci-fication on immunohistochemical staining of osseous and softtissuesrdquo Journal of Diagnostic Pathology vol 7 no 1 pp 45ndash472000

[11] V M Singh R C Salunga V J Huang et al ldquoAnalysis of theeffect of various decalcification agents on the quantity and qual-ity of nucleic acid (DNA and RNA) recovered from bone bio-psiesrdquoAnnals of Diagnostic Pathology vol 17 no 4 pp 322ndash3262013

[12] Y Wang and X Wang ldquoComparison of the application of themodified decalcifying solution and conventional decalcifyingsolution in the bone tissuerdquo Journal of BethuneMilitary MedicalCollege vol 10 no 5 pp 430ndash431 2012

[13] J A Ramos-Vara and M A Miller ldquoWhen tissue antigens andantibodies get along revisiting the technical aspects of immu-nohistochemistry-the red brown and blue techniquerdquo Veteri-nary Pathology vol 51 no 1 pp 42ndash87 2014

[14] R Celiker and S Arslan ldquoComparison of serum insulin-likegrowth factor-1 and growth hormone levels in osteoporoticand non-osteoporotic postmenopausal womenrdquo RheumatologyInternational vol 19 no 6 pp 205ndash208 2000

[15] Y Luo L Cui H Yang and C Fang ldquoThe progress of researchbetween igf-1 and osteoporosisrdquo Clinical Journal of TraditionalChinese Medicine vol 26 no 8 pp 876ndash878 2014

[16] S A Gonzalez-Chavez C Pacheco-Tena C E Macıas-Vaz-quez and E Luevano-Flores ldquoAssessment of different decalcify-ing protocols onOsteopontin andOsteocalcin immunostaining


in whole bone specimens of arthritis rat model by confocalimmunofluorescencerdquo International Journal of Clinical andExperimental Pathology vol 6 no 10 pp 1972ndash1983 2013

[17] Y Guo L Wang R Ma et al ldquoJiangTang XiaoKe granule atten-uates cathepsin K expression and improves IGF-1 expression inthe bone of high fat diet induced KK-Ay diabetic micerdquo LifeSciences vol 148 pp 24ndash30 2016

[18] R Ma R Zhu L Wang et al ldquoDiabetic osteoporosis a reviewof its traditional Chinesemedicinal use and clinical and preclin-ical researchrdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 3218313 13 pages 2016

[19] Y Ying S Wang F Wang et al ldquoThe influence of differentdecalcifying fluid on the section staining of articular jointtissue of rabbit kneerdquoChineseAnimalHusbandry andVeterinaryMedicine vol 39 no 2 pp 221ndash224 2012

[20] N A Athanasou J Quinn A Heryet C G Woods and J OMcGee ldquoEffect of decalcification agents on immunoreactivityof cellular antigensrdquo Journal of Clinical Pathology vol 40 no 8pp 874ndash878 1987

[21] J BMatthews andG IMason ldquoInfluence of decalcifying agentson immunoreactivity of formalin-fixed paraffin-embeddedtissuerdquo The Histochemical Journal vol 16 no 7 pp 771ndash7871984

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ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Hematoxylin-eosin (HE) staining is one of the principalstains in histopathology and the most widely used stain inmedical diagnosis Immunohistochemistry refers to the pro-cess of detecting antigens (eg proteins) in cells of a tissuesection by exploiting the principle of antibodies bindingspecifically to antigens in biological tissues to realize thequalitative and quantitative analysis of antigen [13] Insulin-like growth factor 1 (IGF-1) the most abundant growth factorin bone has the endocrine and paracrine actions during theprocess of bone remodeling [14] In addition IGF-1 positivelycorrelates with bone mineral density in osteoporosis and isimportant for the investigation of bone formation [15]

In order to find the most suitable decalcifying agent weevaluated four different decalcifying solutions for the wholefemurs of adult rats by evaluating the time of decalcifica-tion ease of tissue slicing morphological preservation withHE staining and the preservation of antigenicity for boneproteins such as IGF-1 using immunohistochemical (IHC)staining

2 Materials and Methods

21 Chemicals and Antibody All chemical reagents werepurchased from Beijing Sinopharm Chemical Reagents CoLtd (Beijing China) Rabbit polyclonal IGF-1 antibody wasfrom Santa Cruz Biotechnology Inc (Cat sc-9013 TexasUSA)

22 Animal Feeding Six 16-week-old female Sprague Daw-ley (SD) rats (300 plusmn 10 g) were purchased from ChinaHuafukang Animal Technology Co Ltd (License numberSCXK (Beijing) 2011-0004 Beijing China) Animals werehoused at clean level conditions (certification number SCXK(Jing) 2011-0024) at Beijing University of Chinese Medicine(BUCM) with the temperature of 22 plusmn 1∘C humidity of 55 plusmn5 and a 12 h lightdark cycle All rats were allowed freeaccess to tapwater and foodThe study protocol was approvedby the animal care committee of BUCM China

After one week of acclimation the rats were anes-thetized with pentobarbital sodium (1 sodium pentobarbi-tal 04mL100 g ip) The bilateral femurs were harvestedand stored at minus80∘C for further analyses

23 Decalcification The femurs were prefixed in 4 para-formaldehyde for 24 h Samples were then rinsed in runningtap water for 24 h and incubated with four different decalci-fying solutions (Table 1) (1) 10 EDTA (pH 74) (2) 3 nitricacid (3) 5 nitric acid and (4) 8 hydrochloric acidformicacid followed by neutralizing with 01 aqueous ammoniasolution for 30min Decalcification was performed at 4∘Cunder continuous shaking The decalcifying solutions werechanged on a daily basis and the total decalcification timewasrecorded

The decalcification process was ended when the bonewas easily penetrated through by a needle without any forceSubsequently samples were washed in running tap water for24 h and then followed by routine dehydration and paraffinembedding 5 120583m sections were cut using a Leica microtome

(Leica Germany) and placed on adhesive-precoated (5poly-L-lysine) glass slides

The quality of decalcification was evaluated by the fol-lowing criteria (1) the time of decalcification (2) the ease ofsectioning (3) the morphological preservation by HE stain-ing (4) the antigenicity preservation by IHC staining Theease of sectioning and morphological preservation weregraded from 1 to 4 (1 poor 2 fair 3 good and 4 excellent)[16]

24 HE Staining HE staining was conducted according toroutine protocols [17] Briefly after deparaffinization andrehydration 5120583m longitudinal sections were stained withhematoxylin solution for 5min followed by 5 dips in 1 acidethanol (1 HCl in 70 ethanol) and then rinsed in distilledwater Then the sections were stained with eosin solution for3min and followed by dehydration with graded alcohol andclearing in xylene The mounted slides were then examinedand photographed using an Olympus BX53 fluorescencemicroscope (Tokyo Japan) The staining intensity of thetrabecular bone was analyzed by Image-Pro Plus 60 softwareand expressed as IOD value

25 Immunohistochemical Staining 5 120583m longitudinal sec-tions of the paraffin-embedded femurs were kept at 60∘Cfor 24 h in the oven and then followed by deparaffinizingwith xylene and hydrating with an ethanol gradient (100ndash70) After successively incubating with antigen retrievalsolution (Shanghai Shunbai BiotechnologyCompany Shang-hai China) and 3 H

2O2for 30min the slides were rinsed

with water and incubated with the primary antibody (IGF-1(1 50)) overnight at 4∘C For negative controls the primaryantibody was replaced by nonimmunized serum The nextday the slides were rinsed and incubated with the corre-sponding secondary antibody (Beijing Biosynthesis Biotech-nology Co Ltd Beijing China) for 30min followed by 331015840-diaminobenzidine (DAB) and hematoxylin staining respec-tively The slides were then examined and photographedusing an Olympus BX53 fluorescence microscope (TokyoJapan)TheDAB stainingwas analyzed by Image-Pro Plus 60software

26 Statistics Analysis One-way ANOVA was used to evalu-ate the effect of decalcifying solutions in the quantitative anal-yses of HE staining and IGF-1 antigenicity preservation Sixsections from different rats were taken for histological analy-ses Studentrsquos 119905-test was used to analyze the difference betweengroups A value of 119901 lt 005 was considered significant and119901 lt 001 considered statistically significant

3 Results

31 Time of Decalcification Decalcification in 10 EDTA(pH 74) required 21 days The other decalcifying solutionsneeded 8 days for decalcification with no obvious differencesin the decalcification time




acidformic acid




12

3

(a)

10 EDTA 8 HFA

100x

200x

3HNO3 5HNO3

200 120583m 200 120583m 200 120583m 200 120583m

100 120583m100 120583m100 120583m100 120583m

(b)






4 Discussion







10 EDTA 8 HFA

200x

400x

3HNO3 5HNO3

100 120583m100 120583m100 120583m100 120583m

50 120583m 50 120583m 50 120583m 50 120583m

(a)

020

015

010

005

0HE

stai

ning

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3

lowast

(b)


400x

400x

10 EDTA

8 HFA

3HNO3

5HNO3

50120583m 50 120583m

50 120583m50 120583m

(a)

03

02

01

0

IGF-

1 ex

pres

sion

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3


(b)










Disclosure


Competing Interests




Acknowledgments


References





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




acidformic acid




12

3

(a)

10 EDTA 8 HFA

100x

200x

3HNO3 5HNO3

200 120583m 200 120583m 200 120583m 200 120583m

100 120583m100 120583m100 120583m100 120583m

(b)






4 Discussion







10 EDTA 8 HFA

200x

400x

3HNO3 5HNO3

100 120583m100 120583m100 120583m100 120583m

50 120583m 50 120583m 50 120583m 50 120583m

(a)

020

015

010

005

0HE

stai

ning

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3

lowast

(b)


400x

400x

10 EDTA

8 HFA

3HNO3

5HNO3

50120583m 50 120583m

50 120583m50 120583m

(a)

03

02

01

0

IGF-

1 ex

pres

sion

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3


(b)










Disclosure


Competing Interests




Acknowledgments


References





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





10 EDTA 8 HFA

200x

400x

3HNO3 5HNO3

100 120583m100 120583m100 120583m100 120583m

50 120583m 50 120583m 50 120583m 50 120583m

(a)

020

015

010

005

0HE

stai

ning

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3

lowast

(b)


400x

400x

10 EDTA

8 HFA

3HNO3

5HNO3

50120583m 50 120583m

50 120583m50 120583m

(a)

03

02

01

0

IGF-

1 ex

pres

sion

(IO

D v

alue

)

10

ED

TA

8 H

FA

3HNO3

5HNO3


(b)










Disclosure


Competing Interests




Acknowledgments


References





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






Disclosure


Competing Interests




Acknowledgments


References





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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