We then independent fixed embryos into tubes containing 1015 per tube for immunofluorescence control. If fixed in PFA, rinse embryos in PBS (3 rinses, 5 min. Intro == Histology is definitely a classic technique that is used to study the morphology of cellular and sub-cellular constructions by trimming specimens into thin sections. Even with real-time imaging technology, histological sectioning and staining remain necessary techniques to visualize cellular morphology in cells. Histotechniques are important tools for understanding the progression of cells development and dealing with phenotypes related to mutant backgrounds or pathological claims. Zebrafish are an attractive model for studying cells morphogenesis, owing in part to their optical clarity during embryonic phases. Sectioning through the entire embryo is definitely feasible because embryos are small at embryonic and larval phases (around 1mm)1. Furthermore, bones have not matured at embryonic phases2, so the cells remains soft and provides less resistance when sectioning. Here, we present our optimized protocols for serial sectioning zebrafish embryos with JB-4 plastic resin. Our protocol for embedding and sectioning zebrafish embryos in JB-4 resin (Number 1) has been adapted from your protocol originally offered for JB-4 by Electron Microscopy Sciences and optimized for zebrafish embryos. Work from our laboratory offers used these protocols to analyze kidney and heart development in zebrafish38. We also provide information on how to couple JB-4 embedding with whole-mount RNA in situ hybridization, immunofluorescence and GFP fluorescence. Our protocol can be accomplished in three days as defined inFigure 1. == Number 1. == Timeline of histological protocol using JB-4 resin. == Advantages and Disadvantages of Plastic Embedding == Depending on the type of analysis necessary for a given experiment, there are several histological techniques available, each having their personal advantages and disadvantages. Among the most common techniques are freezing/cryoembedding, paraffin embedding, and plastic embedding for sectioning. For good examples utilizing zebrafish embryos please observe911. Cryoembedded samples do not need to be fixed prior to embedding so sections can be prepared quickly and epitopes are typically well maintained in the sample; however, the cellular morphology is generally poor due to the freezing process. Samples utilized for paraffin embedding require fixation before the embedding process and more cells control methods than cryoembedding; however, morphology is greatly improved. In both systems, immunofluorescence and RNA in situ hybridization can be performed after sectioning. JB-4 is definitely a glycol methacrylate-based polymer used as embedding material that can be used to slice semi-thin or thicker sections. The main advantage to embedding in plastic resins, such as JB-4, is the ability to very easily create ultra-thin (0.51uM) or semi-thin (23um) sections depending on SU 5416 (Semaxinib) the plastic embedding medium12. It has long been appreciated that thin sections provide an increased level of PLXNC1 cytological fine detail over thicker sections. While embedding and sectioning in paraffin SU 5416 (Semaxinib) can provide reasonably good results, semi-thin sections are difficult to produce in paraffin, and the artifacts produced in wax can limit any improvement in cellular resolution that thinner sectioning would provide12. Therefore we recommend our protocol for any analysis where preservation of cellular fine detail is critical. In addition, processing embryos for paraffin embedding requires many more methods and requires dehydration and clearing providers such as xylene which are often toxic and hard to dispose of. While paraffin embedding can be done without special products, it is typically performed using processing machines which minimize exposure to harmful clearing providers, but may not be available to some users, or may process samples inefficiently. By contrast, plastic embedding requires no unique processing equipment, can be performed with or without dehydration, and samples can be stored after embedding indefinitely if processing for morphology. Our protocol is also recommended when sectioning embryos that have previously been processed for RNA in situ hybridization or immunofluorescence, both of which can be altered from SU 5416 (Semaxinib) the multiple methods involved in paraffin processing. While JB-4 resin offers some distinct advantageous in histology, RNA in situ hybridization and immunofluorescence must be performed in wholemount, prior to embedding and sectioning samples. Thus, our protocol is not recommended for analyses where these SU 5416 (Semaxinib) procedures would be more helpful if performed following sectioning. Sample orientation in JB-4 resin can also be demanding because plastic resins are in the beginning water-like, unlike the viscous mediums in freezing and paraffin embedding. But, once we describe with this paper any desired orientation can be achieved utilizing multiple embedding methods. == Experimental Design == Using the protocol described with this paper, we have been successful in embedding and sectioning zebrafish embryos at multiple phases; from as early as 30% epiboly to adults. This protocol is recommended for any analysis where preservation.