Samples preparation
CM, WL
A #10 K-file (Dentsply-Maillefer, Ballaigues, Switzerland) was inserted into the root canals until it was visible under the DOM at the apical foramen to ensure the canal is patent. Length was taken, and 1 mm was subtracted to determine the established working length (WL). A glide path was established. The coronal openings were sealed with a cotton pellet and glass ionomer and the apical foramina were sealed by a double layer of nail polish to prevent their blockage by epoxy resin. The roots were sectioned at 2, 4, 6 mm from the apex—due to the higher prevalence of anastomoses in these levels- using an 0.3-mm-thick Isomet low-speed saw with a diamond blade. The blade was irrigated with IsocutPlus Fluid (Buehler, Lake Bluff, IL, USA) and water according to the manufacturer’s recommendations. The sections obtained were put into an ultrasonic bath with distilled water for 7 min to remove the debris originating from the cut. Pre-instrumentation images of the specimens (IHyflex CM rotary files (Coltène/Whaledent AG, Switzerland) were used to instrument all the root canals up to 35/0.04 taper. A total volume of 5 mL 5.25% NaOCl was used in each canal via a 30-gauge side-vented needle as 1 mL after each file. After complete instrumentation, the sections were disassembled, and images were taken (I