USE OF CERTAIN PROTEIN REAGENTS 



IN THE VAPOUR STATE FOR THE STAINING 



OF REACTIVE CROUPS 



IN FROZEN-DRIED SPECIMENS 



FOR ELECTRON MICROSCOPIC STUDIES 



I. GERSH 



Departrqent pi Anatomy, The University of Chicago, 

 Chicago 37, Illinois, USA 



Thin spécimens (O'I— 0*2 mm) of starved mouse liver were frozen by 

 immersion in propane chilled to — 185X with liquid nitrogen. They 

 were then dried in a vacuum chamber at a température of —40*^ or 

 lower. Without breaking the vacuum the dried spécimens were post-fixed 

 and stained with anhydrous vapors of l-fluoro-2,4-dinitrobenzene (FDNB), 

 and l,5-difluoro-2,4-dinitrobenzene (FFDNB). The former is fluid at 

 room température and pressure, the latter solid. Both reagents react 

 primarily with free amino groups, and probably others, of proteins, 

 polypeptides, and amino acids. The first reagent reacts with such 

 groups singly, while the second reagent forras bridges between neighbor- 

 ing reactive groups of proteins. As both reagents hâve a high molecular 

 weight (186 or more) they cause enhanced contrast in ail adéquate 

 sites where they combine. The structure of the protoplasm is essentially 

 the same as that of unstained frozen-dried material or of frozen-dried 

 material treated with metallic stains. Material post-fixed and stained 

 with FDNB tends to swell during embedding, while material treated 

 with FFDNB shows little or no swelling with the same treatment. 

 Swelling is entirely prevented by double embedding in celloidin and 

 methacrylate. Treatment of frozen-dried spécimens in vacuo with anhyd- 

 rous formaldehyde gas prior to treatment with either reagent reduces 

 the colour and reactivity, and the density in the électron microscope. 

 The implications of thèse findings for an understanding of the term 

 "stabilization" as used in électron microscopy will be discussed. 



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