# Propolis as a Potential Novel Histological Tissue Fixative: A Preliminary Analysis
###### Abstract  
Background: Fixation of biopsy specimens is a critical step before processing and staining them for histological tissue examination. Formalin is considered the gold standard fixative solution for its attributes. However, it has concerning side effects, such as carcinogenic and potential irritational properties. Owing to its various harmful effects, a safer natural substitute should be explored. In this study, we compared the effectiveness of propolisis to 10% formalin and determined its ability as a natural fixative solution. **Materials and Methods:** Sixty tissue specimens were collected from goats' tongues and immediately placed in (1) 6.6% propolisis, (2) 10% natural buffered formalin (positive control), (3) 6.6% propolisis followed by 10% formalin, and (4) 0.9% saline (negative control). Tissue samples were fixed at different time points (12, 24, 48, and 72 h) at room temperature, followed by processing and staining. The quality of the microscopic parameters was blindly assessed by two oral and maxillofacial pathologists using a numerical scoring scale. Scores were statistically analyzed. **Results:** The fixation of tissue samples placed in 6.6% propolisis was statistically significantly better than that of samples placed in 10% formalin and 0.9% saline at different time points. **Conclusion:** Propolis showed promising fixation properties and can be considered a natural alternative to 10% formalin.  
propolis; formalin; tissue fixation; histology +
Footnote †: journal:skin irritation, and eye burning sensation [6; 7; 8]. Moreover, formaldehyde has potential human carcinogenicity that can cause nasopharyngeal cancer, myeloid leukemia, and brain tumors [9; 10]. Therefore, non-toxic natural fixative substitutes should be investigated.  
Propolis, also known as \"bee glue\", is a natural resinous material produced by bees from substances in plants and flower buds [11; 12]. The hue of propolis varies according to the region and the plant it originates from [13]. Its diverse types have a wide melting range of 60-100 \({}^{\circ}\)C. It is rigid at low temperatures and pliable at high temperatures [14]. In contrast to solvents such as water and oil, ethanol rapidly dissolves the primary biologically active components of propolis [15]. Propolis primarily consists of resin (50-70%), oil and wax (30-50%), and pollen (5-10%), as well as chemical elements such as phenol; amino acids; minerals; carbohydrates; vitamins B, C, and E; flavonoids; and carbohydrates [16; 17]. Propolis has numerous biological and pharmacological properties, such as antimicrobial, anti-inflammatory, immunomodulatory, antitumor, and even anti-ulcer effects [18]. These beneficial properties of propolis are attributed to the scavenging activity of free radicals and superoxide anions by butylated hydroxytoluene [19].  
As evident from previous studies, propolis decreases the apoptosis of periodontal ligament cells and can be used as a storage medium for avulsed teeth [20]. However, there is a lack of studies in the available literature regarding the tissue fixation ability of propolis. Therefore, we aimed to assess the fixative properties of propolis, as it is relatively non-toxic and has attractive biological and pharmacological properties, leading us to believe that propolis might be a safer and more effective natural substitute for formalin.