## 4 Discussion
Automated evaluations yielded comparable readouts of slides of the same origin (donor-originated or recipient originated) regarding alveolar circularity and the percentage of alveolar surface area, lectin intensity and SNR of SMA and elastin, whilst the only significant differences were identified between slides of different origins (donor vs. recipient), demonstrating the attributability of such differences to tissue origin (patient factors) instead of alteration of reagents and thus indicating the comparable effects of xylene and isopropanol. Our study indicates a comparable staining effect on LEA, a histochemical marker for type I alveolar epithelial cells, irrespective of whether the tissues were processed via xylene or isopropanol [34, 35, 36]. Furthermore, in the current study, we show that there is no significant difference in the SNR in SMA and elastin antibody stains between xylene and isopropanol. This suggests that these antigen epitopes in lung tissue remain unaffected regardless of whether the lung tissue is processed via either of these two reagents. The ease of tissue sectioning was evaluated for the tissues processed via xylene and isopropanol, revealing no difference in these two groups. Furthermore, this implies the comparable effects of these two clearing reagents on the quality of paraffin and embedding before the sectioning process. As an alternate clearing agent, previous studies have also investigated the viability of coconut oil as a potential alternative for xylene in tissue processing [23, 33, 37]. These studies found no difference in the quality of staining effects, whereas a significantly higher difficulty during the sectioning of coconut oil-processed tissue was observed, thereby limiting the potential of coconut oil as a replacement for xylene [33]. In the process of tissue processing, substantial quantities of xylene are often required for dehydrating and clearing tissues. For instance, our group approximately consumed a total of 43.2 L xylene per year in the large animal experiment. This large volume of xylene must then be safely discarded, as the inappropriate disposal of waste can result in a detrimental environmental pollutant, posing potential hazards to human indirectly [38, 39]. Therefore, finding and using alternatives to xylene for tissue processing in histological research has a profoundly positive impact on health and the environment. Due to its widespread application and the potential for adverse effects resulting from exposure to xylene, it is currently listed in the European Chemical Agency's \"Community Rolling Action Plan (CoRAP)\" [40]. Studies show that short-term exposure to xylene results in irritation of the respiratory mucosa, which may lead to lung edema [41]. Furthermore,the symptoms of acute dermal exposure to xylene includes skin erythema, dryness, and scaling [42], whereas prolonged exposure to low concentrations of xylene may lead to hazardous effects on the respiratory and cardiovascular system, central nervous system, digestive system, reproductive system, and renal system, eventually resulting in chronic, long-term health problems [13; 14; 41; 42; 43; 44]. Consequently, in consideration of user safety, isopropanol emerges as a less toxic and harmful alternative for the human body. It proves to be more environmentally sustainable, economically friendly, and affordable [45]. It is noteworthy that technological advancements have ushered in the standardization of automated tissue processors in most laboratories, addressing the need to reduce operators' exposure to chemical reagents. This development not only markedly enhances the efficiency of experiments but also aligns with the broader objective of minimizing potential bio-hazardous effects associated with chemical reagents. Moreover, the consideration of isopropanol as a viable alternative clearing reagent to replace xylene reflects a strategic move toward safer practices.