All the samples were stored in saline solution at 37°C for 48 hours to accomplish complete setting of the sealer. After 2 days, all the study specimens were thoroughly dried. The roots of all the experimental groups and the positive control group were coated with two coats of nail varnish except for the apical 2 mm. The entire root surface and apical foramen of the negative control specimens were completely covered with two coats of the nail varnish. The specimens were allowed to dry completely, then placed in 2% methylene blue dye solution (Central Drug House, New Delhi, India) and centrifuged at 3,000 rpm for 5 minutes. Afterwards the teeth were rinsed under tap water to remove the dye on external tooth surfaces.