# An Easy-to-Use Histological Technique for Small Biological Samples of Senegalese Sole Larvae
## 4 Discussion
The method of pre-embedment in agarose gel before sectioning minimized the difficulty of handling extremely small sized samples. Although literature already had documented the pre-embedding technique in agar since the 1960s for small tissue biopsies, this is not a routine technique in worldwide histopathology laboratories [44], especially in environmental studies. Some authors have applied this technique for studying zebrafish larvae [5; 24; 25], but, to our knowledge, this is the first time that the pre-embedding agar is used in Solea larvae. The advantages of using molecular biology grade agarose for embedding small biological samples include the softness for a more accessible block section, the substance hardness similar to the fixed sample, and the positive impact on orientation before tissue sectioning. This technique is also inexpensive, safe, and easy to use [44], and this agarose embedding process is applicable for the examination of any small sized organisms or tissues [5] such as fish, molluss, annelids, crustaceans, and others, commonly used for ecotoxicological assessment purposes. In this study, the histological methods for fixation, agarose pre-embedding, paraffin embedding, sectioning, staining and digital image capture (Figure 1) were appropriate for flatfish larvae. It is helpful to note that some stiffer samples must be decalcified (after fixed) for adequate sectioning. For example, the fixed samples can be immersed in 7% EDTA (15 min; 60 \({}^{\circ}\)C) or 10% nitric acid (6 h) [45], and then proceeded to pre-soak in agarose. Even though, nowadays, the histology practice is mainly restricted to specialized automated labs that enable a quicker and more standardized workflow, some steps within the process are still entirely dependent on the manual performance, accuracy and capability of laboratory personnel [44]. Previous works have designed a method to embed fish larvae producing simultaneous large-scale sections on a single glass microscope slide [5; 24]. However, it is preferable to individually embed samples because it requires more specialized staff and proper equipment to produce adequate large-scale sections. Figure 4: Photomicrographs of 8-day-old _Solea senegalensis_ larvae applying the easy-to-use histological technique developed in this study. (**A**) Hyperaemia (*) in hepatic tissue at 1.00 mg Cu-Al LDH-L\({}^{-1}\) exposure. (**B**) Vacuolation of hepatocytes (arrow) at 0.10 mg PE-L\({}^{-1}\) + 3.33 mg Cu-Al LDH-L\({}^{-1}\). (**C,D**) Gastrointestinal deformity and dilation (arrowhead) at 0.10 mg PE-L\({}^{-1}\) + 3.33 mg Cu-Al LDH-L\({}^{-1}\) and at 1.00 mg PE-L\({}^{-1}\) + 3.33 mg Cu-Al LDH-L\({}^{-1}\). H&E staining. Scale bar 20 \(\upmu\)m.  
The proposed protocol involves old practices such as pre-impeding in agarose and replacing complicated laboratory instruments with simple equipment and materials such as aluminum foil. The successful adaptation alternatives represent economic and easy-to-handle advantages. This proposed protocol draws the attention of researchers interested in the field as it is possible to make histological sections of good quality with simple means and resources, provided by basic equipment, such as fume hoods, laboratory drying ovens, microtomes, and microscopes. It can also be helpful in research areas or institutes that do not have well-equipped laboratories and encourage researchers to devise methods that enable them to take advantage of the materials and tools available.