## 1 Introduction
has proven that the use of Rhodamine B can lead to the dye being leached from the sealers [16]. This in turn may provide the distorted, inadequate and unrepresentative measurements of the sealer penetration made under CLSM compared to SEM-based measurements. The aforementioned paper by [PERSON] et al. concluded with the words \"Staining of sealers using a fluorescent dye, such as rhodamine B, is an inadequate method for the evaluation of the sealers' penetration depth into dentinal tubules\", and a strong clinical need to develop and evaluate both new types of sealers and protocols for sealing dental tubules became an inspiration for the authors to do researchwith the use of two dyes characterized by different properties in order to minimize the research error. In this paper, the authors made the conscious decision to use fluorescein and porphyrin dyes to minimalize the potential error and to check if the type of dye used to label the sealer affects the results. Fluorescein was used to stain the sealers in previous studies with the use of the CLSM by [PERSON] et al. and [PERSON] et al. [17; 18]. The probability that the hydrophilic fluorescein dye may leak from the sealer has been taken into account and is indicated as a weakness of the study listed in a special discussion paragraph on the possible shortcomings of the studies presented. To avoid abovementioned weakness and compare the obtained more likely results, the decision to use a second, hydrophobic dye was made. In a previous study carried out by the authors, hydrophobic porphyrin (5,10,15,20-Tetraphenyl-21H,23H-porphine (TPP)) was used to stain the sealer [19]. In contrast to the previous study, in this experiment hydrophobic porphyrin was used to verify if the results obtained with both dyes were comparable. In theory, the hydrophilic fluorescein might leach more in an aqueous environment and the hydrophobic porphyrin may leak into the alcohol in the tubules. These results show that the use of porphyrin alone may be somewhat imprecise and is another shortcoming of the present study. Since the need to study the penetration of sealants into dental tubules is still strong, the authors, aware of the weaknesses of currently available testing methods, used both hydrophilic and hydrophobic dyes to optimize the current research results as much as possible. The novelty of the study is the evaluation of a modified irrigation protocol during root canal treatment in terms of penetration of sealants into the dental tubules as well as the attempt to look for alternative dyeing methods used to stain the sealers. The clinical relevance of this paper is concerned with the effect of the last irrigating solution on sealer penetration into the tubules. In a typical clinical scenario, it is very probable that not all the bacteria/microbiota would be killed and flushed away by using sodium hypochlorite. The added tubule closure with the sealer might therefore be beneficial to entrap the remaining debris/microorganisms in the tubules. It is clinically important to know which of the final irrigants is more favorable prior the obturation with the hydrophobic epoxy sealer in terms of sealer's penetration into the dentine tubules. The null hypothesis in the presented study is that the use of alcohol in the final phase of irrigation of the root canals with sodium hypochlorite will enhance the depth of penetration of the epoxy sealer into the dentinal tubules. The aim of this study was to compare the penetration depth of the epoxy sealer into the dentinal tubules after performing three irrigation protocols.