Enzyme Clearing of Alcian Blue Stained Whole Small Vertebrates for Demonstration of Cartilage

Abstract

Preparation of small vertebrates cleared after alcian blue Staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formalin, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess db blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately we-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alixarin red S in 0.5% KOH. Specimens rue bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which me routinely fixed in formalin and stored in alcohol.