CHAPTER XXXIL 433 



sharper, but increases the length and cost of the method, and may 

 cause precipitates to form, especially where much myelin is present. 

 (3) It is possible to keep sections, which cannot be stained imme- 

 diately, for some days or even a fortnight, in redistilled water 

 to which a few drops of formalin have been added. Thorough 

 washing with redistilled water is then imperative before they are 

 transferred into the 2 per cent, silver nitrate solution. (4) Sections 

 of nervous tissues must not remain in the 2 per cent, silver 

 nitrate more than forty-eight hours, or precipitates may form. 

 The longer their stay there, the longer must be the washing 

 before staining ; this, however, must not, as a rule, exceed five 

 minutes. (5) The volume to which the ammoniacal silver nitrate 

 is diluted should be 35 to 45 c.c., and the sections remain in it fifteen 

 to twenty minutes. The subsequent washing before transferring 

 the sections into 20 per cent, formalin should not occupy more than 

 ten to fifteen seconds, and their stay in the final formalin solution 

 (especially for cerebral cortex) should not exceed two to three hours. 



The other eight Da Fano modifications (Proc. Physiol. Soc., 

 Journ. Physiol., liii, 1919-20) were all proposed for the study of 

 cortex cerebelli, and are characterised by a special treatment of 

 the sections (cut by the freezing method) with various reagents 

 before transferring them into the 2 per cent, silver nitrate solution, 

 nothing having been changed, however, in regard to the long fixation 

 of material in formalin and -the use of redistilled water. They may 

 be summarised as follows : 



Mod. 3. Place sections, after washing in redistilled water, in 

 2 to 3 per cent, silver nitrate at 36 to 37 C. for about twenty-four 

 hours ; wash quickly ; stain in ammoniacal silver nitrate solution 

 diluted to 40 c.c. for thirty minutes. Wash, reduce, tone, and 

 mount as usual. 



Mod. 4. Place sections in 50 per cent, pyridine for six to eighteen 

 hours ; wash in redistilled water for twenty-four to forty-eight 

 hours ; 2 per cent, silver nitrate at 37 C. for twenty-four hours, etc., 

 as in Mod. 3. 



Mod. 5. Place sections in pure pyridine for four to twelve hours. 

 Wash in redistilled water overnight. Transfer sections into 20 per 

 cent, formalin prepared with redistilled water for about twenty-four 

 hours. Wash again in redistilled water overnight ; 2 per cent, silver 

 nitrate at 37 C., etc., as before. 



Mod. 6. Sections are treated first with 20 per cent, formalin, 

 and then with pure pyridine, in the reverse order of Mod. 5. 



Mods. 7 and 8. The same as Mods. 5 and 6, but replacing the 



M. 28 



