Stains and Staining 81 



Regaud's Mordant Hematoxylin Stain: 



This solution is the best of the general-purpose "iron hematoxylins" and 

 should be widely used to stain the nuclei before complex afterstaining. 

 The staining solution must be ripened for some time before use. The most 

 convenient method of doing this is to prepare a 10 per cent solution of 

 hematoxylin in alcohol and then to dilute this with the glycerin in water 

 immediately before use. The alcohol solution of hematoxylin should be at 

 least a month old. This stain is very slow if it is used cold, so that it is 

 customary to heat both the mordanting and staining solutions to about 

 50° C before use. 



Sections Are Stained as Follows: 



1. Accumulate the sections in distilled water. 



2. Transfer the slides to the mordanting solution for 30 min at 50° C or 

 overnight at room temperature. 



3. Rinse each slide in distilled water to avoid carrying over too much 

 of the mordant into the staining solution. 



4. Transfer the slides to the staining solution for 30 min at 50° C or 

 overnight at room temperature. 



5. Transfer the slides to distilled water and wash until no more stain 

 comes away. 



6. Dip each slide up and down in the differentiating solution until it 

 appears to be partly differentiated and transfer to tap water until no 

 further color comes away. Then examine the slide under the microscope. 

 If further differentiation is required, repeat the process. 



7. Transfer all the slides to tap water until they have turned blue. If the 

 tap water becomes yellow from traces of picric acid, it should be changed 

 or differentiation will continue. Hematoxylin is very sensitive to acids and, 

 as it comes from the differentiating solution, has a reddish-brown color, 

 as distinct from the clear blue color required on the finished slide. In 

 many parts of the world, natural tap water is sufficiently alkaline to pro- 



