## 2 Materials and Methods  
### 2.1. Teeth Collection, Preparation, and Distribution into Groups  
Based on a previously established experimental model [2], 70 single-rooted, freshly extracted human teeth were kept in 0.05% sodium hypochlorite liquid and were selected for the experiment. The experiment was approved by the Tel-Aviv University Ethics Committee on 27 March 2018, and all methods were implemented according to the relevant regulations and guidelines. All included teeth were fully developed and presented with a single straight root canal (curvature of \(<\)5 degrees) [9]. The following teeth were excluded: teeth in which the ratio of long to short canal diameters was \(>\)2 (\"long oval canals\") [10], root canals without apical patency or with an apical diameter of \(>\)#25 K-file, previously endodontically treated teeth, and teeth with root resorption. The crowns of the included teeth were cut, and 13-mm-long root specimens were obtained. The working length was assessed by a #15 stainless steel K-file (K-file; Dentsply Maillefer, Tulsa, OK, USA) extending beyond the apical foramen. The root canals were prepared to apical diameter #30 with standard hand K-files (K-file; Dentsply Maillefer, Tulsa, OK, USA) by the \"balanced force technique\" [11]. Copious irrigations were performed during the instrumentation by 5% sodium hypochlorite solution. Eventually, final irrigations were done in order to remove the smear layer (17% EDTA followed by 5% sodium hypochlorite). Then, [PERSON] high-speed burs (Zakaria; Mailleffer, Ballaigues, Switzerland) were used to cut the apical 3 mm of the root apices without bevel. Three-millimeter retrograde preparations were made using diamond-coated ultrasonic retro-tips (AS3D tip; Satelec, Paris, France) [2]. Paper points were used to dry the prepared retrograde cavities. Before the retrograde filling, a standard gutta percha cone was adjusted 3 mm short of the root apex in order to control the retrograde filling depths. To avoid sealer contamination between the retrograde material and the dentin, no sealer was used.  
In order to allow for analysis under CLSM, each retrograde filling material was mixed in accordance with the manufacturer's instructions and was fluorescently labeled by adding dye (Alexa Fluor 350 dye; Life Technologies, Carlsbad, CA, USA) at an estimated ratio of 0.1% (weight) during mixing [6].  
The filling was carried into the retrograde cavities by carriers (Dovgan Carriers; Quality Aspirators, Duncanville, Texas).  
The prepared samples were then randomly allocated into eight experimental and control groups:  
1. (\(n\) = 15): Retrograde 3 mm preparations filled with fluorescently labeled MTA (ProRoot; Dentsply Tulsa Dental, Johnson City, TN, USA).
2. (\(n\) = 15): Retrograde 3 mm preparations filled with fluorescently labeled IRM (IRM; Dentsply, Mannheim, Germany).
3. (\(n\) = 15): Retrograde 3 mm preparations filled with fluorescently labeled Biodentine (Biodentine; Septodont, Saint-Maur-des-Fosses, France).
4. (\(n\) = 5): Retrograde 3 mm preparations, not filled (Positive control).
5. (\(n\) = 5): Teeth without retrograde preparation or filling. The whole external root surface including the apical foramen was sealed with 2 layers of nail varnish (Negative control) [12; 13].
6. (\(n\) = 5): Like Group 1 but without ensuing bacterial contamination (MTA negative control).
7. (\(n\) = 5): Like Group 2 but without ensuing bacterial contamination (IRM negative control).
8. (\(n\) = 5): Like Group 3 but without ensuing bacterial contamination (Biodentine negative control).  
All specimens were then stored at 100% humidity and 37 \({}^{\circ}\)C for 24 h to allow for setting of the retrograde materials.