## 2 Materials and Methods  
A sample size of 10 per group was calculated with G*Power 3.1.4 (Kiel University, Kiel, Germany) to set the study power at 80%. Seventy-two human single-root teeth, each with an intact crown and fully formed apex, were kept in 0.01% NaOCl solution after extractions for periodontal disease. The soft tissues, bone fragments and cement were removed from the root surface using a hand curette and Sof-lex discs (3M ESPE, Seefeld, Germany). After the access opening, the canal scouting and the initial glide path were performed with a size 10 K-File at the working length (WL) usingGlyde (Dentsply Sirona, Switzerland) as a lubricating agent. The mechanical glide path was performed with ProGlider (Dentsply Sirona, Switzerland) (tip size = 0.16 mm, taper = 0.02 up to 0.85 mm). Root canal shaping was completed with ProTaper Next X1, X2 and X3 (PTN X3 tip size = 0.30 mm, taper = 0.07 mm). The specimens were irrigated by alternating 5% sodium hypochlorite (NaOCl) and 10% ethylenediaminetetraacetic acid (EDTA) to a total of 10 mL of each solution per specimen. A 4 mm long root dentin block was horizontally sectioned for each tooth, starting 1 mm below the cement-enamel junction, with a 0.6 mm thick precision diamond saw (Isomet 5000; Buehler Ltd., Lake Bluff, IL) at 1000 rpm, under water cooling. Afterwards, the root canals' diameters were standardized to the size of the Gates-Glidden drill no. 4 (1.1 mm in diameter) (Dentsply Sirona, Ballaigues, Switzerland), at 300 rpm, under water cooling. All of the dentin blocks were immersed in 5% NaOCl and 17% EDTA, each for 15 min, in an ultrasonic bath, to remove any organic matter and smear layer, then rinsed with sterile water for 10 min. The previous procedure was also performed to minimize the fluorescence from the dentin. The roots were inspected under a microscope to verify the canal cleanliness and the absence of cracks. The specimens were stored in saline prior to their packaging and sterilization in an autoclave. The sterilized specimens were then placed in a multi-well test plate under a laminar flow biohazard cabinet (CLANLAF--VFR 1206, Racine, WI, USA). The root canals were infected with an overnight culture of _Enterococcus faecalis_ ATCC 29212 at a turbidity of \(3\times 10^{7}\) colony forming units/mL, as confirmed by the colony counts in triplicate, in Brain Heart Infusion broth (BHI; Oxoid, Milan, Italy). _E. faecalis_ ATCC 29212 was chosen for this study after a careful review of the literature, which revealed that this bacterium has multiple properties that lead to its key role as an endodontic pathogen [32]. The samples were incubated under aerobic conditions at 37 \({}^{\circ}\)C for 3 weeks and were centrifugated to allow for the penetration of _E. faecalis_ into the dentin tubules. The broth was replaced every 4 days [33] and the culture purity was checked regularly. After 3 weeks, the specimens were randomly assigned to 3 experimental groups (each group N = 20), positive (N = 6) and negative (N = 6) control groups. The specimens in the first experimental group were exposed to the 3-MIX antibiotic paste (ciprofloxacin 2 ug/mL, metronidazolezole 8 ug/mL and minocycline 4 ug/mL). Those in the second experimental group were exposed to the 3-MIXC paste (ciprofloxacin 2 ug/mL, metronidazolezole 8 ug/mL and clarithromycin 2 ug/mL), and the specimens in the third group were exposed to the 2-MIX (ciprofloxacin 2 ug/mL and metronidazolezole 8 ug/mL) antibiotic paste. The different antibiotic formulations were mixed with MG or HA as vehicles, dividing each experimental group in two sub-groups (N = 10). Experimental samples were exposed to antibiotics for three weeks [11; 24], with fresh broth every 4 days. Twelve specimens served as controls: six teeth were autoclaved and used as negative controls, while 6 teeth did, after bacterial incubation, not receive canal disinfection, were inoculated with HA or MG and were used as positive controls. These samples were used to assess the biofilm formation through the CLSM examination (Figure 1).