JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 16:81-82 (1990)

Rapid Communication IMPROVED URANYL ACETATE STAINING FOR ELECTRON MICROSCOPY

Bernard Tandl e r Department o f Oral Biology, School o f Dentistry, Case Western Reserve Uni v e r s i t y , C1 eve1and , Ohio 44106 Uranyl acetate was one o f the f i r s t contrasting agents t o be used t o s t a i n t h i n sections f o r e l e c t r o n microscopy (Watson, 1958). Because t h i s compound i s only sparingly soluble i n water, absolute methanol, i n which very high concentrations o f uranyl acetate can be achieved, has been advocated as a Unfortunately, both aqueous and methanolic solvent (Stempak and Ward, 1964) solutions o f uranyl acetate are i n h e r e n t l y unstable, becoming t u r b i d and unusable s h o r t l y a f t e r preparation. I n order t o f o r e s t a l l t h i s photoactive r e a c t i o n o f uranyl acetate solutions, many workers s t o r e such solutions i n brown b o t t l e s o r wrap c l e a r f l a s k s i n aluminum f o i l t o keep out l i g h t , but, even w i t h these precautions, the s t a i n i n g solutions s t i l l may p r e c i p i t a t e . (.

I have devised a simple method f o r s t a b i l i z i n g aqueous uranyl acetate solutions and a s t a i n i n g method f o r t h i s agent t h a t i s v i r t u a l l y foolproof. An amount o f uranyl acetate s u f f i c i e n t t o guarantee s a t u r a t i o n ( % 1.5 - 2 g) i s placed i n a clear, 60 m l , glass-stoppered b o t t l e , the b o t t l e i s f i l l e d w i t h The uranyl d i s t i l l e d water, and one drop o f g l a c i a l a c e t i c a c i d i s added. acetate i s allowed t o dissolve overnight, and i s then ready t o use. The a d d i t i o n o f a c e t i c a c i d t o t h e uranyl acetate s o l u t i o n renders i t stable i n d e f i n i t e l y - - i t can be kept i n the l i g h t f o r months a t room temperature w i thout p r e c i p i t a t i ng

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To use t h i s s o l u t i o n i n a s t a i n i n g sequence, a small a l i q u o t i s f i l t e r e d through a s i n g l e sheet o f No. 43 o r 44 Whatman f i l t e r paper i n t o a small, glass-stoppered graduated cylinder. A f t e r an equal amount o f absolute methanol i s added and mixed by i n v e r t i n g t h e closed c y l i n d e r several times, the r e s u l t a n t mixture, which should be used fresh, i s ready t o use. A f t e r the g r i d s t h a t are being stained have f l o a t e d section side down atop t h e s t a i n i n g s o l u t i o n f o r an appropriate i n t e r v a l (time determined by inspection-- usually 10-20 minutes), they are picked up and washed. For e f f e c t i v e cleaning o f t h e grids, they i n i t i a l l y are exposed t o a stream o f 50% methanol from a s q u i r t b o t t l e . The stream i s n o t d i r e c t e d a t the g r i d i t s e l f , since t h i s a c t i o n can rupture the sections, b u t i s played on the meniscus formed by the apposed t i p s o f the forceps grasping the grid-- s t a i n i n g s o l u t i o n retained i n t h i s meniscus o r d i n a r i l y i s a primary source o f s t a i n contamination. Washing i s then continued w i t h d i s t i l l e d water. The uranyl acetate s t a i n i n g i s followed by s t a i n i n g i n the usual way w i t h any desired lead solution, as per M i l l o n i g (19611, Reynolds (19631, o r Venable and Coggeshall (19651, or w i t h bismuth subni t r a t e (Riva, 1974). A1 though g r i d s stained i n t h i s manner occasionally may show 1ead contamination , they almost never exhi b i t contamination traceable t o uranium. 6 1990 WILEY-LISS, INC.

Received May 11, 1990; accepted May 18, 1990

B. TANDLER

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To increase contrast, many workers expose t i s s u e specimens en bloc t o uranyl acetate, which then acts as a mordant f o r f u r t h e r s t a i n i n g o f t h i n An sections. Acetic a c i d can be used t o s t a b i l i z e such s t a i n i n g solutions. aqueous s o l u t i o n o f uranyl acetate i s s t a b i l i z e d by adding one drop o f g l a c i a l a c e t i c a c i d t o 100 m l o f solution. The recomnended concentration f o r uranyl Higher concentrations work well f o r acetate f o r en b l o c s t a i n i n g i s 0.25%. increasing e l e c t r o n contrast, b u t i n t e r f e r e w i t h t o l u i d i n e o r methylene blue s t a i n i n g o f semithin sections meant f o r l i g h t microscopy. To s t a i n en bloc, the t i s s u e specimens e i t h e r are f i x e d i n i t i a l l y i n o r are p o s t f i x e d i n osmium tetroxide. The specimens are r i n s e d i n several changes o f d i s t i l l e d water, then soaked overnight i n the r e f r i g e r a t o r i n 0.25% uranyl acetate t h a t has been s t a b i l i z e d w i t h a c e t i c acid. The t i s s u e i s once again r i n s e d i n several changes o f d i s t i l l e d water, then i s dehydrated and embedded by conventional means. These s t a i n i ng protocol s , which employ a c e t i c acid-stabi 1ized uranyl acetate, y i e l d tissues o f high c o n t r a s t t h a t almost always are f r e e o f uranium contamination; t h e s t a i n i n g solutions themselves have an extended s h e l f - l i f e . REFERENCES M i l l o n i g , G. (1961) A modified procedure f o r lead s t a i n i n g o f t h i n sections. J B i ophys. Biochem. Cytol , 11 :736-739.

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Reynolds, E.S. (1963) The use o f lead c i t r a t e a t high pH as an electron-dense s t a i n i n e l e c t r o n microscopy. J. C e l l Biol., 17:208-212. Riva, A. (1974) A simple and r a p i d s t a i n i n g method f o r enchancing the c o n t r a s t o f ti ssues p r e v i ously stained w i t h uranyl acetate. J M i croscopi e,

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19:105-108.

Stempak, J.G., and Ward, R.T. (1964) An improved s t a i n i n g method f o r e l e c t r o n J . C e l l B i o l , 22: 697-701. microscopy

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and Coggeshall, R. Venable, J.H., (1965) A s i m p l i f i e d lead c i t r a t e s t a i n f o r use i n e l e c t r o n microscopy. J. C e l l Biol., 25(No. 2, P t . 1):407-408, Watson, M.L. heavy metals.

(1958) Staining of t i s s u e sections f o r e l e c t r o n microscopy w i t h J . Biophys. Biochem. Cytol ., 4:475-478,

Supported i n p a r t by N I H grant 07648.

Improved uranyl acetate staining for electron microscopy.

JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 16:81-82 (1990) Rapid Communication IMPROVED URANYL ACETATE STAINING FOR ELECTRON MICROSCOPY Bernard Tandl...
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