# Propolis as a Potential Novel Histological Tissue Fixative: A Preliminary Analysis
## 4 Discussion  
Formalin (10%) is the leading tissue fixative agent worldwide for histology and research laboratories. However, owing to the serious and harmful side effects of formaldehyde-based solutions such as 10% formalin, other superior and safer alternative natural fixative agents should be explored and researched. In this study, propolis displayed promising results in terms of tissue fixation at all time points. Propolis composition may play a role in its effectiveness as tissue fixative and its superiority over 10% formalin. Propolis is composed mainly of a mixture of resin (40-50%), wax (25-30%), essential oils (10%), pollen (5%), and organic compounds such as steroids, amino acids, and polyphenols [24]. The stickiness of propolis may protect tissue samples from the invasion of various pathogens (putrefaction) and reinforce cellular structure integrity [24]. Furthermore, the presence of a mixture of resin and wax in the propolis composition could contribute to the crosslinking of tissue molecular proteins, which accelerates and enhances its fixation ability. Organic compounds, by their scavenging action, prevent tissue putrefaction and autolysis due to their antimicrobial and antioxidant effects, and this may also improve propolis fixation properties [19]. This could explain why the tissue samples that were placed in propolis were firmer during grossing and fixed faster at 12 h and improved over time when compared to 10% formalin, where its fixation capacity improved after 24 h.  
To the best of our knowledge, no study in the current literature has been conducted to investigate the fixative properties of propolis. However, several other investigations have been conducted to find alternative natural solutions to be used as substitutes for formalin. For example, honey has demonstrated promising fixative properties [25]. Sugar syrup and jaggery have also shown encouraging results as alternative natural fixative agents [8]. [PERSON] et al. conducted an experimental in vitro study on five commercially available fresh goat meats (buccal mucosa) that were placed in 20% buffered formalin, distilled water, 20% honey, 20% sugar syrup, and 30% jaggery syrup. After storing the tissues for 24 h, they reported that honey, sugar, and jaggery syrup fixation ability were similar to each other and closer to formalin, with jaggery syrup showing the best results [26]. These results are consistent with the findings from our study where honey, sugar, and jaggery syrup can be used as alternative natural fixative agents. However, our study showed that propolis had superior fixation ability compared to 10% formalin after 24 h. The same research group conducted another study to evaluate the fixation properties of 20% honey and 30% jaggery  
Figure 8: Photomicrographs of a hematoxylin-eosin-stained section of lower 1/3 rd of epithelium at 48 h (40\(\times\)): (**A**) 0.9% saline; (**B**) 10% formalin; (**C**) 6.6% propolis; (**D**) 6.6% propolis followed by 10% formalin. sympt over 6 months and concluded that jaggery and honey demonstrated satisfactory results after 6 months, and jaggery was comparable to formalin in tissue fixation [27]. [PERSON] et al. investigated the effectiveness of honey as a natural substitute for formalin by fixing kidney tissue and rat liver in honey and formalin for 24 h; they found that tissues fixed in low honey concentrations (10% and 20%) had comparable results to formalin-fixed control tissues, and increased concentrations of honey produced slower penetration rates and increased tissue hardening, making them difficult to section [28]. A similar study was conducted by [PERSON] et al., who tested the fixation and preservation efficacy of 10% processed and unprocessed honey for 24 h compared to 10% formalin. All groups had an adequate fixation for diagnosis, as there were no statistically significant differences in the scores between the groups. However, they found that the presence of staining artifacts was higher in the honey groups.