Counterstaining of Nauta-Gygax Impregnated Sections with Cresyl Violet

Frozen sections of formalin-fixed brains are impregnated according to the Nauta-Gygax method. After treatment in a 1% Na2S2O3 solution, the sections are washed thoroughly, then bleached for 10–20 sec in a solution prepared by diluting 1 part of a stock solution consisting of: borax, 2 gm; potassium ferricyanide, 2.5 gm; distilled water, 100 ml, with 4 parts of distilled water, washed well, transferred to 1% Na2S2O3, and washed again. The loose sections are placed in Carnoy's 6:3:1 fluid for 5 min, then floated onto glass slides which have been coated with 1:1 albumen-glycerol, and drained and dried at room temperature. The bleached sections affixed to the slides are stained 30 min in 0.1% cresyl violet adjusted to pH 3.6–3.8 by adding 10% acetic acid. The sections are differentiated and dehydrated in ascending grades of alcohol and cleared in 2 changes of xylene, and then covered in balsam.