JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 1:205-206 (1984)

PROCESSING W L TISSUE SPECIMENS IN ACRYLIC RESINS FOR ULTRAMICROTOWY: IMPROVED HANDLIWG AND ORIENTATION

R i c h a r f i L. Ridgway and Ya t thew H. C h e s t n u t , Depar tment o f ? o o l o g y and Department o f Botany, Washington S t a t e U n i v e r s i t y , Pullman, WA 99164

INTROD11CTION

O b t a i n i n g t h i n p l a s t i c sec t i ons o f a s p e c i f i c t i s s u e reg ion f o r l i g h t and/or e l e c t r o n microscopy r e q u i r e s t h a t p r e c i s e o r i e n t a t i o n o f t he specimen be ma in ta ined throughout . t h e embedding p rocess . Methods i n t r o d u c e d t o a c h i e v e such o r i e n t a t i o n have been des igned p r i m a r i l y f o r use w i t h epoxy res ins . Gain ing i n p o p u l a r i t y , however, a re a number o f water-misc ib le low v i s c o s i t y a c r y l i c r e s i n s (e.g., g l y c o l methacrylate, L.R. White, e tc . ) hav ing p o t e n t i a l a p p l i c a t i o n s i n immunocytochemical , enzyme h is tochemical , and X-ray m i c r o a n a l y t i c a l s t u d i e s . W P present . h e r e a mathod f o r p r o c e s s i n g s m a l l t i s s u e specimens i n a c r y l i c r e s i n s t h a t combines t h e w e l l known technique o f agar pre-embedding fsee S c o t t -- e t a l , 1970) w i t h t h e use o f a p o l y e s t e r s u p p o r t p l a t f o r m . The disc-shaped p l a t f o r m serves as a specimen c a r r i e r d u r i n g p r o c e s s i n g and, t o g e t h e r w i t h t h e agar s u p p o r t , a i d s i n m i n t a i n i n g specimen o r i e n t a t i o n d u r i n g r e s i n po lymer izat ion. A f t e r t he r e s i n i s cured, removal of t he d i sc p rov ides a p r e d i c t a b l e p l a n a r s u r f a c e r e l a t i v e t o the specimen, which f a c i l i t a t e s t h e t r imming of b lock faces.

MATERIALS 4ND METHODS

P o l y e s t e r s u p p o r t p l a t f o r m s 3 re prepared by f i r s t develop ing a sheet o f unexposed photographic f i l m t o remove i t s emu ls ion , and t h e n p u n c h i n g d i s c s f r o m t h i s m a t e r i a l u s i n g a standard h o l e punch. We have used Kodak e l e c t r o n image f i l m $4499 (ESTAP. t h i c k b a s e l , b u t o t h e r f i l m s m i g h t work e q u a l l y w e l l . The d i s c s are f i n i s h e d by making s h o r t p a r a l l e l c u t s i n t o one edge wi t .h a r a z o r b lade , and f o l d i n g t o form a tab as shown i n F igu re 1 A . The tab serves as bo th a re ference p o i n t f o r o r i e n t a t i o n and a h a n d l e f o r specimen t r a n s f e r . The d i s c s lapprox. 7 mm diam.) f i t i n t o s i z e +!I0 g e l a t i n capsules. For use i n pre-embedding, a 27 ag r s o l u t i o n i s prepared and kep t l i q u i d i n a stock b o t t l e nraintained i n a 45 8C water ba th u n t i l needed.

No d e v i a t i o n from normal pr imary f i x a t i o n procedures i s necessary. I n ou r laborator ies,pr imary f i x a t i o n i s u s u a l l y done i n s i t u , and i s f o l l o w e d by s e v e r a l b u f f e r r i n s e s . A f te rward , small t i s s u e specimens are c u t from t h e f i x e d m a t e r i a l under a d i s s e c t i n g microscope, and b l o t t e d c a r e f u l l y w i t h f i l t e r paper t o prevent. subsequent d i l u t i o n o f t he agar. Each specimen i s then p o s i t i o n e d on i t s own support d i s c w i t h i n a f r e s h l y p i p e t t e d d r o p l e t o f 27 agar ( F i g . 1B). A f t e r t h e des i red o r i e n t a t i o n i s achieved r e l a t i v e t o t h e d i s c , t h e a g a r d r o p l e t i s l e f t t o g e l !2-5 min. ) . The s u p p o r t e d specimen ( a g a r d r o p l e t p l u s d i s c ) i s t h e n p l a c e d i n t o a v i a l c o n t a i n i n g b u f f e r s o l u t i o n , w h i l e o t h e r samples a re brought t o t h i s stage.

I f osmium t c t r o x i d e o r o the r secondary f i x a t i o n i s d e s i r e d , i t can be c a r r i e d o u t a t t b i s t i m e . A l t e r n a t i v e l y , such f i x a t i o n can be done j u s t p r i o r t o aga r pre-embedding. The specimens a r e t h e n d e h y d r a t e d i n an ,e thano l s e r i e s and i n f i l t r a t e d s t e p w i se (acco rd ing t o t h e manufacturers ' i n s t r u c t i o n s ) i n the approp r ia te s o l v e n t / a c r y l i c r e s i n m i x t u r e s . W h i l e no i n c r e a s e s i n i n f i l t r a t i o n t i m e have been f o u n d necessa ry , t h e use o f a

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Received December 16, 1983; accepted December 20, 1983.

0 1984 ALAN R. LISS, INC.

206 R.L. RIDGWAY AND M.H. CHESTNUT

r o t a r y i n f i l t r a t i o n d e v i c e i s suggested. F o l l o w i n q two changes o f p u r e r e s i n , each specimen i s p o s i t i o n e d h o r i z o n t a l l y i n a r e s i n - f i l l e d g e l a t i n c a p s u l e ( F i g . 1C) w h i c h i s t h e n c a p p e d a n d h e a t c u r e d . A f t e r p o l y m e r i z a t i o n , g e l a t i n capsules a re d i sso l ved i n w a r m water and each r e s i n b lock i s t r imned down t o the l e v e l o f t he p o l y e s t e r support d isc. The d i sc i s then removed wi th forceps t o reveal t h e o r i e n t e d specimpn j u s t helow t h e cleavage p lane (F ig . ID). Block faces c o n t a i n i n g the a rea o f i n t e r e s t can then be trimmed r a p i d l y and accu ra te l y p r i o r t o sec t i on ing .

FIGURE 1.

disc 6 specimen

agar

cap

k n

gelatin capsule

specimen

plane

resin block

A B C

COMMENTS

S i n c e p o l y m e r i z a t i o n o f a c r y l i c r e s i n s is i n h i b i t e d by atmosphpric oxygen, s t a n d a r d embeddinq p r o t o c o l s i n t e n d e d f o r epoxy r e s i n s must b-e m o d i f i e d t o p roduce a c r y l i c b l o c k s o f o p t i m a l q u a l i t y f o r s e c t i o n i n g . G e l a t i n capsules are favored over po l ye thy lene molds, which tend t o Droduce b locks o f non-uniform hardness. However, n e i t h e r the g e l a t i n capsules alone n o r c u r r e n t l y used f l a t embedding methods f o r a c r y l i c r e s i n s (Feder and O'Br ien, 1968; G r e n v i l l e , 1993) can ensure t h a t p rec i se o r i e n t a t i o n o f s m a l l t i s s u e specimens i s v a i n t a i n p d d u r i n g r e s i n p o l y m p r i z a t i o n . Those methods a l s o p r o v i d e l i t t l e p r o t e c t i o n f o r d e l i c a t e t i s s t i p s a q a i n s t qechanical damage that may occur du r ing processing. Whi le t h e use o f photographic f i l m as a specimen s u p p o r t i s n o t new ( s e e Makinen and 4 r s t i l a , 10!55), i t s a p p l i c a t i o n a s d e s c r i b e d h e r e i s unique. The present method represents a cons iderable improvement i n t h e hand l i ng and o r i e n t a t i o n o f specimens du r ing a c r y l i c r e s i n embedding.

REFERENCES

G r e n v i l l e , D. (1993) A new m e t h o d f o r f l a t e m b e d 4 i n g i n p l y c o l

Feder, N., and O ' B r i e n , T.P. (la69) P l a n t microtechnique: Yome p r i n c i p l e s

Makinen, E., and A r s t i l a , A . ( 1 9 6 5 ) O r i e n t a t i o n o f t i s s u e h l o c k s

methacrylate. S t a i n Technol ., 58: 57-58.

and new methods. Am. J. 9ot., 55: 123-142.

u l t r a t h i n s e c t i o n i n g -- A one-stage embedding procedure. 40: 373.

S t a i n Technol.,

S c o t t , K., T a r i n , D., and Sharp, J.A. (197C) g r i e n t a t i o n o f s p h e r i c a l specimens f o r u l t r a t h i n s e c t i o n i n g i n s e l e c t e d p l a n e s by embedding i n agar. J. Microscopy, 91: 217-220.