## 2 Materials and Methods
### Sample Selection and Endodontic Procedures  
The sample size was calculated considering previous research related to tubular penetration.  
Forty-two human single-rooted premolars extracted because of suffering periodontal disease were selected with the following exclusion criteria: roots with root caries or not completely formed root, canals with calcite metamorphosis, fissures or fractures, root resorptions, previously treated teeth and canals presenting double curvature, and the presence of more than one canal. Periapical radiographs of each tooth in buccolingual and mesio-distal directions were made to confirm the presence of one canal.  
Using an ultrasonic tip, calculus was removed from all the roots and stored for one day in a 2% thymol solution. Subsequently, they were decorated to achieve a canal size of 12 mm in length. A 08 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was inserted into the canals until it was visualized in the apical foramen. To determine the working length, this length was reduced by 0.5 mm. After the insertion of a 10 K-file (Dentsply Maillefer, Ballaigues, Switzerland), each canal was instrumented with the files of the basic sequence of the Blue Shaper(r) system (Zarcdendo, Gijon, Spain) up to the working length at 500 rpm (4.0 Ncm). The Eighteeth E-Connect S motor (Town, Xinbei District, Changzhou City, Jiangsu Province, China) was used. Irrigation with 2 mL of 5.25% NaCl (masterful formulation) between files was performed with a Monojet 27G syringe (Cardinal Health 7000 Cardinal Place Dublin, OH 43017, USA) at 2 mm away from the length where the needle is embedded. Patency of the canals was ensured during all the treatments. Each file was used in 3 canals. To create a closed system, the apical third of the teeth was blocked with Reus wax [11].  
Three mL of 5.25% NaOCl and one mL of 17% EDTA were used and both solutions were activated in three 20-s periods renewing the irrigant between them. A final wash was performed with 5.25% NaOCl. This activation was completed with the Ultra X ultrasonic activator (Eighteeth, Jiangsu) at a frequency of 45 kHz using the blue tip (25/02) (Eighteeth, Jiangsu) at one less millimeter than the working length inside the canals.  
The canals were dried with 25/02 paper points (Dentsply Maillefer, Ballaigues, Switzerland). Then, using a 25 verifier (Dentsply Maillefer, Ballaigues, Switzerland) an apical calibration was performed. Samples with an interior diameter larger than #25 were discarded and replaced with another tooth. After apical calibration, the teeth were differentiated into two groups depending on the fluorescent staining (Rhodamine B or FLuo-3). Two teeth were used as the control group.  
Twenty teeth in the first group were obturated performing the single-cone technique and using TotalFill BC Sealer(r) mixed with the fluorescent stain Rhodamine B (Alquera Ciencia S.L, Cadiz, Spain) at a concentration of 0.1%. In the second group, a further 20 teeth were filled using the single-cone technique and TotalFill BC Sealer(r) mixed with Fluo-3 (Invitrogen, Carlsbad, CA, USA) fluorescent stain at a concentration of 0.1%. In both groups, the single-cone technique was performed with Autofit 06 gutta-percha calibrated to 0.25 mm with a gutta-percha calibrator ([PERSON], Ballaigues, Switzerland).  
One control tooth was finally irrigated with 1 mL of deionized water in which Rhodamine B was dissolved at a concentration of 0.1%, and the other control tooth was irrigated with 1 mL of deionized water in which Fluo-3 was dissolved at a concentration of 0.1%. Both control teeth were dried briefly to avoid excess moisture. All concentrations were calculated using a microbalance.  
All filled canals were sealed with Cavit(r) (3M ESPE AG.Dental Products, Seefeld, Germany). The samples were radiographed to check the obturation and stored at 37 \({}^{\circ}\)C and 100% humidity.