## 4 Discussion  
The primary purpose of tissue processing is to efficiently replace water content present in the tissues with another medium, enhancing tissue support and solidification [24]. In histology laboratories, the most frequently utilized clearing reagent for tissue processing is xylene, as it facilitates paraffin infiltration [2; 25]. Nevertheless, it is well established that xylene poses potential hazards to the health of researchers and the environment [18; 26]. There is sufficient evidence supporting significant toxicity with exposure, which has widely provoked the desire for optimization [17; 18; 27]. Efforts to achieve this purpose led to proposals for several clearing agents of less toxicity, better environmental friendliness,  
Figure 4: Multiplex antibody labeling efficacy of lung tissue in xylene and isopropanol clearing. (**a**) Representative images of bronchioles stained for SMA and elastin, as well as lectin and DAPI, from a tissue sample cleared with xylene. (**b**) Representative images of bronchioles stained for SMA and elastin, as well as lectin and DAPI, from a tissue sample cleared with isopropanol. Scale bar in the pictures represents 100 \(\upmu\)m. (**c**) Quantification of the SNR between xylene and isopropanol tissue clearing for an SMA primary antibody stain with a 568-secondary conjugate. (**d**) Quantification of the SNR between xylene and isopropanol tissue clearing for an elastin primary antibody stain with a 647-secondary conjugate. \(n=6\). DAPI, \(4^{\prime}\),6-diamidino-2-phenylindole; LEA, Lycopersicon esculent lectin; SMA, smooth muscle actin. SNR, signal-to-noise ratio. and higher cost-effectiveness, like isopropanol, which facilitates paraffin infiltration and the procedures thereafter. This study confirmed that the staining and sectioning of the tissue processed via isopropanol were comparable to that via xylene in human lung tissue samples [28, 29, 30, 31, 32]. Previous studies have been conducted comparing various tissue processing reagents, yet few were conducted in a systematic manner with objective evaluation methods, which was particularly notable in the context of lung tissue [32, 33]. In light of this, our study employed a systematic comparison of the staining effects of xylene and isopropanol on lung tissue, adopting both H&E and immunofluorescence staining methods in our research. Both blinded observation and the evaluation of H&E staining indicated the comparable overall quality of slides processed via xylene and isopropanol. This is in keeping with previous studies conducted by [PERSON] et al. [22] and [PERSON] et al. [32] who both identified no significant difference between slides processed via xylene and isopropanol in kidney, liver, stomach and adenocarcinoma samples; however, their studies lacked systematic evaluation as well as the inclusion of lung tissue. Our study employed routine lung tissue assessments for a more comprehensive and thorough comparison of the possible different effects the two processing methods might introduce to final readouts. Although seemingly comprehensive, popularly applied H&E histological evaluations are mainly based on subjective notions relating to observer knowledge and proficiency, producing risks of introducing potential and inevitable bias especially if implemented alone as in some of the previous studies. Therefore, the incorporation of fluorescent staining, both molecular and antibody-based, coupled with automated confocal and Ti2 microscopic imaging and analysis, was conducted here to diminish human-based limitations.