India Ink-Gelatin Vascular Injection of Skeletal Tissues

Dogs under sodium pentothal anesthesia were given 100 mg of heparin intravenously and, 15 min later, exsanguinated. A mixture consisting of Higgins India ink #4415, 1 part, and a 14% solution of calfskin gelatin, 1 part, was injected (at 37° C) into the femoral artery of a disarticulated hindlimb. A pump-type 5 ml hypodermic syringe was connected with a gravity-feed reservoir that contained the injection mass, with a filter in the outflow from the reservoir. Veins severed by the disarticulation of the limb permitted a free flow of the injection fluid out of the vascular tree. After injection of about 400 ml of fluid by pumping the syringe, the limb was chilled (—20° C) to solidify the gelatin. Specimens from cleaned bones were sawed out and fixed in Zenker-formalin solution for 24 hr, then decalcified with 6% sulfosalicylic acid. The technique was completed by embedding in celloidin and sectioning. Blood vessels in both cortical and trabecular regions were completely filled by the injection mass.