164 Microscopic Histochemistry 



other interesting observation is the fairly intense staining of 

 both myeloid granules and cell nuclei with peroxide-free 

 acidified solutions of benzidine, prepared with tap water 

 ( eflPect of free chlorine? ) . 



These facts clearly indicate that the oxidation of various 

 substrates by peroxides can be promoted by more than one 

 mechanism; in fact, even the invariable involvement of per- 

 oxides in what is called a "peroxidase reaction" is not certain. 

 The clarification of this problem will require much more 

 investigative work. 



In histochemistry the most important application of the 

 peroxidase reaction is the demonstration of hemoglobin. 

 After hemolytic reactions, hemoglobin retains its enzymatic 

 properties ( in renal tubules ) for only 24-36 hours, although 

 its other staining reactions remain unchanged for several 

 days. 



Methods 



Smears are fixed with acetone, alcohol, or formalin-alcohol 

 (1:10). For tissues the same fixatives or formalin-saline are 

 recommended. It is important that the fixative should not 

 hemolyze the red cells, thereby causing a diffusion of the 

 reaction for hemoglobin. Frozen sections, are the best, al- 

 though in most cases excellent results are obtained after 

 celloidin- or paraffin-embedding. 



A) Lisons'^^ zinc-leuco method gives the best results for 

 fresh hemoglobin. Prepare the following staining solution: 

 Dissolve 1 g. of either acid fuchsin, acid violet, or patent blue 

 in 100 ml. of a 2 per cent acetic acid solution; add 5-10 g. 

 of zinc dust, and boil mixture until the dye is decolorized 

 (patent blue will bleach only to a pale brown). Add 2 more 

 ml. of concentrated acetic acid. Keep the mixture in the ice- 

 box. In the case of recolorization, heat briefly until decolor- 

 ized once more. For use, filter 10 ml. and add 1 ml. of com- 

 mercial (3 per cent ) hydrogen peroxide to it. Pour mixture 

 over the slide. Hemoglobin will stain very intensely almost 



