## 2 Materials and Methods
### Preparation, Irrigation and Filling of the Root Canals  
The root canals were negotiated using C-pilot files (VDW, Munich, Germany), size ISO#10. The working length was set by reaching the anatomical foramen and then subtracting 1 mm. After the root canals were prepared with Reciproc 25 and 40 instruments (VDW) using Silver Reciproc micromotor (VDW) and calibrated with ISO K#40 files (VDW), after which the patency was tested (recapitulation) using the C-pilot ISO#10 file. During the preparation, a lubricant (FileCare, VDW) was used on every instrument. After using each file, the root canals were rinsed with 1 mL of 5.25% sodium hypochlorite. After the instrumentation of the root canals, the apexes were closed using wax in order to simulate the periapical tissue resistance (a closed system). Then the roots were randomly allocatedinto 3 groups (n\({}_{1}\) = 30). Afterwards the root canals were irrigated according to the following irrigation protocols: \begin{tabular}{l l} \hline Group 1: & 5.25\% sodium hypochlorite--120 s--5 mL \\ \hline Group 2: & 5.25\% sodium hypochlorite--30 s--1 mL \\  & 40\% citric acid--30 s--1 mL \\  & 5.25\% sodium hypochlorite--30 s--1 mL \\  & 40\% citric acid--30 s--1 mL \\  & 5.25\% sodium hypochlorite--60 s--2 mL \\ \hline Group 3: & 5.25\% sodium hypochlorite--30 s--1 mL \\  & 40\% citric acid--30 s--1 mL \\  & 5.25\% sodium hypochlorite--30 s--1 mL \\  & 40\% citric acid--30 s--1 mL \\  & 5.25\% sodium hypochlorite--60 s--2 mL \\  & isopropyl alcohol--60 s--2 mL \\ \hline \end{tabular}   
In groups 2 and 3, the smear layer was removed by alternating irrigation with 5.25% sodium hypochlorite (NaOCI) and 40% citric acid (CA). In previous studies performed by the authors, this method of smear layer removal was the most effective [22,23]. In group 2, 5.25% NaOCI was used as the final irrigant, and in group 3 isopropyl alcohol was used as the last fluid. The liquids were applied to each canal using a needle (size 0.4 \(\times\) 19.0 mm) in 0.5 mL portions. The needle was inserted with up-and-down movements up to 1-2 mm shorter than the working length. Subsequently, each liquid was passively activated with ultrasounds for 5 s using the spreader ISO 35 (VDW) with the E1 tip of a Smart Piezo scaler (Mectron, Carasco, Italy). During the spreader activation up-and-down movements were performed, limited to the maximal depth 1-2 mm shorter than the working length [8,24]. Each cycle of fluid exchange and activation lasted 15 s, thus the stages that lasted 30, 60 and 120 s were carried out in 2, 4 and 8 exchange-activation cycles, respectively. After irrigation, the root canals were dried using paper points (VDW). Following that, the roots from all of the groups were randomly allocated into two equal subgroups (F and P), each containing 15 roots (n\({}_{2}\) = 15). The subgroups differed in the type of marker added to the sealer. In F subgroups, the sealer was modified by adding a saturated alcohol solution of fluorescein to obtain its concentration of 0.1%. In the studies carried out by [PERSON] et al. and [PERSON] et al., fluorescein was added to the sealers to achieve 0,1% concentration [17,18]. In P subgroups, the sealer was modified using saturated alcohol solution of porphyrin to obtain a concentration of 0.1%. In both subgroups, excess alcohol was evaporated from the sealer by blowing dry air at room temperature. The same protocol was used in the previous experiment carried out by the authors, which proved to be an effective way of introducing the dye into the epoxy sealer [19]. The root canals were filled with gutta-percha with marked epoxy sealer AH Plus (Dentsply, Bensheim, Germany) using the thermoplastic vertical condensation technique with the B&L SuperEndo (B&L, Jeonju, Republic of Korea). The 0.4 taper gutta-percha cones were calibrated to ISO 40 size using the gutta-percha tip gauge A0186 ([PERSON] (Ecublens, Switzerland), Dentsply).