CHEMICAL METHODS • 13 



experiment is required if all substances pre-existing in the tissue and 

 capable of giving a positive reaction can be removed without seri- 

 ously reducing the enzyme activity. This has been accomplished 

 in certain tissues for the phosphatase test. 



Many, if not most, of the tests described in the following pages 

 leave much to be desired. In some cases they have been developed 

 for particular tissues and cannot be adapted to others without a 

 certain amount of additional research. Most of the tests are clearly 

 not at all good. However, it is the purpose of the writer to present 

 the published methods for the localization of substances, groups, 

 and enzymes, even though they may be, and usually are, poor. In 

 this way the investigator who requires a particular method will 

 have at hand the procedures already developed, and, if they should 

 prove inadequate, at least he will have them as a basis from which 

 to work out improvements. 



A word should be added concerning the mounting media employed 

 for tissue sections. The media given in the procedures that follow 

 are those used by the original authors. However, newer media are 

 available and they may be substituted for the balsam or damar 

 that have been employed in the past. A 60% solution of Clarite in 

 xylol appears to be superior to neutral balsam for mounting sections 

 since, according to Lillie (1941), Clarite does not promote the 

 fading of some stains to the degree that balsam does. Stowell and 

 Albers ( 1943) showed Clarite absorbs less visible light than balsam. 

 Tetrachloroethylene may also be used as a solvent for Clarite. 

 Clarite and Clarite X (also called Nevillite V and Nevillite No. 1, 

 respectively) are superior in all respects to balsam and damar, 

 according to Groat ( 1939) . A solution of 60% of the resin in 40% 

 of toluol by weight is recommended. The resins are clean, cheap, 

 water-white, inert, high-melting, absolutely neutral, and chemically 

 homogeneous. Clarite X undergoes a slight yellowing with age and 

 has a refractive index of 1.567 while Clarite is very color stable and 

 has a refractive index of 1.544. 



The limited availability of certain reagents or enzyme substrates 

 may make it imperative to employ a considerably smaller volume 

 than is normally used in staining dishes and Coplin jars. The 

 hanging-drop technique (Glick and Fischer, 1945a) may be adopted 

 in these cases. The section on the slide is surrounded by a circle 

 of vaseline or stopcock grease, a drop of the reagent or substrate 



