Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.