A B S T R A C TThis paper describes a simple protocol of paraffin-embedded histological section for fish eggs, embryo and larvae of the hard-lipped barb and the giant gourami. The specimens were fixed in Bouin solution, washed in 70% ethanol, then were dehydrated in a series of ethanol solution of increasing concentration until absolute ethanol was reached. The specimens were cleared in graded xylene and were infiltrated with liquid paraffin then were embedded in pure paraffin. Upon sectioning, at 4-5 µm thick the specimens were attached to the gelatin-coated glass slide and let to dry at room temperature or 37°C overnight. The specimens were deparaffinized in xylene, rehydrated then were stained with hematoxylin and eosin. After being dehydrated in graded ethanol, the specimens were cleared in xylene and were mounted with an organic mounting agent. Any step in preparing histological section including samples collection, fixation, dehydration, infiltration and embedding might contribute to the quality of histological features. A proper knowledge of the tissues beeing processed, fixative solution and the histological techniques is essential to gain good results. Bouin fixative is preferable to fix fish larvae and produce a good histological feature. Decalcification is necessary to produce a good histological section on the specimens containing bone.
KEY WORDS: paraffin embedded, fish embryo, fish larvae, Osteochilus vitatus, Osphronemus goramyCorresponding Author: Gratiana E Wijayanti | email: 1.bugrat@gmail.com
I N T R O D U C T I O NThe most routinely employed techniques of histological preparation is the paraffin embedded sections. Since it first publication by Professor Edwin Klebs in early 1860s (Sanderson et al., 2013), paraffin embedded sections had served as an important tool in the field of biomedical (Grave, 2007;Treuting et al., 2012) as well as developmental biology (e.g. Balogh and Sótonyi, 2003;Habashy et al., 2012;Mathew and Appel, 2016). Using histological examination scientists can detect a stable condition or early sign of diseases not easily recognized by a morphological examination. The paraffin-embedded section in combination with immunoreaction has developed into a powerful technique to study tissue lineage and fate map, the immunohistochemistry.The paraffin-embedded technique has been widely used in fish research to study general structure of various organs as well as their pathological examination (Takashima and Hibiya, 1985;Palikova et al., 2004). Protocols for processing various fish organs are readily available for example eyes (Nuckel and Gross, 2007), gills, liver, kidney (Carmago andMartinez, 2007) and gonad (Wijayanti et al., 2009). However, paraffin embedded protocol for egg, embryo, and larvae are less. One of the most comprehensive protocols for fish larvae was developed by Sabaliaukas et al. (2006) enabling to process a large number of zebrafish larvae at the same time. This protocol, however, needs more sophisticated equipment such as acrylic mold and automated-closed reagent...
