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http://jdr.sagepub.com/Journal of Dental Research

http://jdr.sagepub.com/content/45/4/1237.citationThe online version of this article can be found at:

DOI: 10.1177/00220345660450044701

1966 45: 1237J DENT RESEdward J. Coleman and Salvatore J. Desalva

Rapid Decalcification for Histochemistry

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International and American Associations for Dental Research

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R a p i d D e c a l c i f i c a t i o n f o r H i s t o c h e m i s t r yEDWARD J . COLEMiN f a n d SALV[TORE J . D E S ' A L TAD e p a r t m e n t o f P h a r m a c o l o g y , C o l g a l t e - a h l o l l i e R e s e a r c h C e n t e r , N e u w , B r u n s -w i c k , N e t J e r s e y

When h i s t o c h e m i c a l l o c a l i z a t i o n o f e n z m n e s i s t o b ed o n e on t i e c a l c i f i e 1 h a r d t i s s u e s , i t i s i m p e r a t i v e t op e r f o r m t h e d e c a l c i f i c a t i o n i n t h e s h o r t e s t t i m e p e -r i o d p o s s i b l e i n o r d e r t o p r e s e r v e a s much o f t h eenzyme m a s s e s a s p o s s i b l e i n t h e i r o r i g i n a l s t a t e .Even u s i n g o n e o f t h e more e f f i c a c i o u s l e l c a l c i f x - i n g ra g - e n t s , E " I ) T A j * i t was n e c e s s a r y t o p r o v i d e somet h i n g o n t h e o r d e r o f 4 8 h ) u r s o f d e c a l c i f i c a t i o n ( K .B A t o G H s , J . d e n t . R e s . , 1 2 : 4 5 7 - 6 6 , 1 9 6 3 ) . W h i l e s u c ht r e a t m e n t a l l o w e d g o o d h l i s t o c h e r n i c a l l o c a l i z a t i o n s ,a n unknown amount o f t h e enzyme w a s s t i l l l o s t a n dmay h a v e c a u s e d a n i n c o m l ) l e t e e n z xy m e p i c t u r e .The c u r r e n t s t u d y o f f e r s a m e t h o d o f r e l a t i v e l yg e n t l e d e c a l c i f i c a t i o n i n a much s h o r t e r t i m e p e r i o e l .A 1 0 p e r c e n t s o l u t i o n o f EDTA\ was p s r e p l a r e d li n 0 . 1 M p h o s p h a t e b u f f e r a t p ) H 7 . t ) , a n d t h e f i n a lpH a d j u s t e d w i t h NaOH t o 7 . 0 . F r e s h , u n f i x e l b o n es i e c i n e n s x e r e ) r e l ) a r e d i x - d i s s e c t i n g a x x a v a l l s o f tt i s s u e . B o n e s l ) e c i m e n s a s l a r g e a s an e n t i r e h a m s t e rs k u l l ( 2 5 - m m . - l o n g a x i s ) o r t h e e n t i r e m a x i l l a r y J a x xc u t a x a v f r o m t h e s i o l l l e a v i n g o n l x r t h e m o l a rt e e t h h a v e b e e n u s e I .

Revend f o r p u b l i c a d i o n O t o b e r 1 8 , 1 d 9 ; .* J d r s u t r w t a m i n u d e d i .1 e d i t d i f .

U s i n g an ' " o n i c B o n e l ) e c a l c i f i e r , " t t h e s p e c i -m e n s s e r e i l a c e 1 i n a r e c e p t a c l e a n t i s u b m e r g e d i na p p r o x i m a t e l y 4 0 0 c c . o f t h e EDTTA s o l u t i o n . T h i sa p p a r a t u s i s c o m p l e t e l y s e l f - c o n t a i n e d a n d i s i np r o p e r o p e r a t i o n w h e n b u b b l e s a re s ee n t o r i s e f r o mt h e g r a p h i t e a n c d p l a t i n u m e l e c t r o d e s . The b o n eS p e c i m e n s n o t e d a b o v e w e r e c o m p l e t e l y e l e c a l c i f i e di n l e s s t h a n 1 2 h o u r s .The E J ) I D T s o l u t i o n w a s s t i r r e d c o n s t a n t l y a n dh e l i x l i e l o - i N r 1 0 ' C . t o p r e s e r v e t h e e n z y m e c o j i t e n t .I ) e c a l c i f i c a t i o n w x a s c h e . . k e t i b x i a l i a t i o n a n dn e c l l e - p r o ) b i n g o f t h e s p e c i m - e n s . T h e s p e c i m e n s w e r et h e n f r o z e n i n a l i q u i d p r o t e i n m a t r i x a t - 2 0 C .o n a c r x o s t a t t a n d I s e c t i o n e d a t 1 0 g u . The s e c t i o n sw e r e t h a w e a t r o o m t e m p e r a t u r e , a n d t h e c h o s e nh i s t o c h e m i L a l l i r o c e c l u r e x a s u n d e r t a k e n , o r t h es e c t i o n s x x e r c i l a c e d l d i r e ~ t l x i n t o 5 0 p e r c e n t g l u -t a r d l d l e h v l e f o r f i x a t i o n . E i t h e r m e t h o d l a l l o w c c l e x -c e l l e n t h i s t o c h e m i c a l e n z ' s m e l o c a l i z a t i o n s , a l t h o u g hl e t t e r c e l l u l a r d e t a i l was a c h i e v e d l b v t h e l a t -t e r i l l u s t r a t i o n .

M a r t i n S w e e t st o . , L o u i s v i l e t Kl ITt. l n c r u i m e n i t s ( C o , V i e s t m i n t ! 1 1 1 .

S e c t i o n s o f h a m s t e r m a x i l l a r v t h i r d m o l a r . L e f l : 35X o f t o o t h ; c e n t e r : l O O X o f o n e c u s p ) . s h o x i n g p r e s e r v a -t i o n o f p u l p c e l l s a n d c l e a r l y d e f i n e d c i e n t i n a l t u b u l e s : r i a i h t : 1 , O O O X o f t h e d l e n t i n . s h o o t i n g e n z x me a c c u m u -l a t i o n s w i t h i n t h e d e n t i n a l t u b u l e s , i . e . , i n t h e p r o t o p l a s m o f t h e o d o n t o b l a s t i c p r o c e s s e s .

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