Specific Protocol for Diaphanization and Bone Staining in Sceloporus grammicus (Tschudi, 1845)

Abstract

Introduction: Diaphanization, a specialized histological process that utilizes chemical substances such as potassium hydroxide (KOH), allows for the clarification of soft tissues without affecting hard tissues like bones and cartilage. This is of vital importance in anatomical, ecotoxicological, and comparative studies. Materials and Methods: Heuristic trials were conducted, adjusting the concentrations proposed by Romero and González (2019) for the fish Gymnocorimbus ternetzi, specifically for Sceloporus grammicus. Two specimens were used, each with different chemical and temporal concentrations. Subsequently, the transparency of each was analyzed using the opacity histogram with the distribution of values in the alpha channel, with the aim of finding significant differences through a Student's t-test. Results: A statistically significant difference in transparency between specimens A and B was identified (t = 4.5017, p < 0.0001). Lizard B showed a considerably higher average transparency (211.92) compared to Lizard A (157.71). The 95% confidence intervals, both parametric and bootstrap, strongly supported the importance of this disparity. Conclusions: Adapting existing techniques, as in this study, is crucial to optimize results and ensure applicability. The variation between eviscerated and intact specimens highlights the dilemma of balancing transparency and structural integrity. The rapid transparency in the eviscerated specimen may cause fragility, while the technique in the intact specimen is more gradual but potentially more effective in structural conservation. Given the lack of specific protocols for each species, this study contributes to the knowledge of histological techniques.