CHAPTER II 



THE SPECIAL FEATURES OF HISTO- 

 CHEMICAL METHODS 



The usual requirements of a satisfactory reaction in ana- 

 lytical chemistry are specificity and sensitivity. In histochem- 

 istry, since one of the main objects is accurate localization, 

 two more conditions must be fulfilled. 



First of all, the chemical substances to be identified must 

 be immobilized at the sites they have occupied in the living 

 tissue. This is no problem as long as substances such as cal- 

 cium phosphate, hemosiderin, lipids, etc., insoluble in well- 

 chosen fixatives, have to be demonstrated. It is a relatively 

 minor problem in the case of large and poorly diflFusible 

 molecules, like proteins and glycogen. These are, as a rule, 

 precipitated by the fixative or emmeshed inextricably in a 

 spongework of other co-precipitated substances before any 

 gross displacement can take place. It must be remarked, 

 however, that minor shifts on a cytological scale are not nec- 

 essarily prevented. As the fixative penetrates into the interior 

 of the tissue, the advancing front of a high-concentration 

 gradient (especially when a fixative which acts partly by 

 dehydration, such as alcohol or acetone, is used) may push 

 certain substances ahead of itself until they are stopped by 

 an impermeable barrier, such as a cell membrane. In this way 

 artifacts of the type of the well-known "glycogen flight" may 

 be produced. They are usually most marked near the surface 

 of the tissue block, where the tissue is hit by a sudden high 

 concentration of the fixative. 



In the case of easily soluble and highly diffusible sub- 

 stances (ions, sugars, ascorbic acid, urea, etc.), the regular 

 methods of fixation cannot effect an immobilization, even 



10 



