# An Easy-to-Use Histological Technique for Small Biological Samples of Senegalese Sole Larvae
## 4 Discussion  
In this work, we described an accessible protocol (summarized in Table 3 and detailed in Section 2) to apply histopathological techniques in small sized organisms by deploying standard laboratory equipment. The use of fresh ethanol and xylene solutions is essential to ensure processing quality [25]. Despite the fixation process preserves the specimens over time, we recommend the histological processing of the samples within a maximum of 6 months after fixation to avoid the degradation of the sample, such as excessive dryness and distortion of the tissues. In the end, the paraffin blocks were kept at \(-20\)\({}^{\circ}\)C for a few minutes before cutting, avoiding slice curling. Soaking the blocks in ice water before their sectioning produced optimal sections, as advised by [PERSON] et al. [25]. Another suggestion is that although the sections were stretched over the flame of a Bunsen burner glass alcohol lamp, it is safer to use a water bath (at approximately 40 \({}^{\circ}\)C) lightly sprinkled with colorless gelatin (to ensure that the paraffin slices firmly adhere to the slides), always taking care not to melt or overstretch the floating sections. The slides produced according to the proposed method were efficient for the histological analyses. Finally, the photomicrographs can be registered with photographic equipment (e.g., a cell phone fitted to the microscope's eye lens), and the frequency and occurrence of the histopathological alterations can be identified and classified into an index, as proposed by [PERSON] et al. [7] and [PERSON] et al. [21]. Considering the development phase of the analyzed organism, the digestive organs (mainly the liver and intestine) and the epidermis are the target organs for histopathological biomarkers responses. These organs enclose histopathological alterations that can be semi-quantitative indexed in different reaction patterns, including circulatory disturbances (e.g., hemorrhage, hyperemia, and aneurysm), regressive changes (implying functional loss, e.g., nuclear alterations, atrophy, and necrosis), progressive changes (involving altered function, e.g., hypertrophy, vacuolation, and hyperplasia), inflammatory responses (e.g., melanomacrophage centers and lymphocytic infiltration), and tumors [7; 17]. In a recent study by [PERSON] et al. [17], flatfish _S. senegalensis_ larvae were assessed in terms of histopathological and biochemical effects upon exposure to polyethylene microplastics (PE) and copper-aluminum layered double hydroxides (Cu-Al LDH), both single and combined exposure. In fact, [PERSON] et al. [17] reported the first case-study based on the histological technique here fully detailed and explored. Additionally, Figure 4 shows the frequent histopathological responses observed in the _S. senegalensis_ larva due to the mentioned exposure. [PERSON] and co-authors [17] demonstrated that larvae exhibited a notable deformity in the intestinal tract caused by microplastic ingestion (Figure 4C,D), along with low frequencies of histopathological lesions, mainly at the liver, including hyperaemia (Figure 4A,D) and hepatocyte vacuolization (Figure 4D). The evidence of mild inflammation, reversible as the exposure ends [32], reinforces the use of histopathological biomarkers in Senegal sole larvae. Such findings demonstrated how valuable histopathology is for more integrative and holistic assessments in ecotoxicology. \begin{table}
\begin{tabular}{c c} \hline
**Expensive, Specialized Equipment** & **Alternatives** \\ \hline Automatic sample processor & Swap solutions manually \\  & Laboratory fume hood \\ Automatic sample preparation system & Swap solutions manually \\ embedding centers & Laboratory fume hood \\ Mold for embedding cassettes & Handmade box-shaped aluminum sheets \\ Histopathology water bath & Bunsen burner glass alcohol lamp \\ Microscope camera & Smartphone camera \\ \hline \end{tabular}
\end{table} 
Table 3: Low-cost and easy-to-use alternatives for the histological processing.