staining of harvested zebrafish scales

Colheita e visualização de escamas elasmóides de peixe-zebra durante a regeneração in vivo

Bone is a hard connective tissue that forms a major part of the skeleton, enabling locomotion and acting as a mineral reserve in the body. In order to maintain healthy bone, an exquisite balance between bone formation and degradation is essential via the coupled activity of osteoblasts (which are anabolic) and osteoclasts (which resorb bone). This balance is disrupted by aging or hormonal imbalance, often leading to bone fragility diseases such as osteoporosis1. Although existing drugs have been approved to target bone fragility diseases, many have side effects; therefore, there is a need for new therapeutics1. Thus, there remains a need for abundant sources of biologically relevant bone tissue that can be used to test potential therapeutic compounds.
Traditionally, rodent models and cell culture systems have been used to study bone biology. However, zebrafish are increasingly becoming another model of choice. While not a mammalian system, zebrafish offer certain advantages for bone research over rodents; these include their fecundity and the translucency of the larvae; even in adulthood, some skeletal tissues, including the scales and the fins, remain translucent, allowing high-resolution in vivo imaging and the increased availability of skeletal mutants2,3. Both the fins and scales of zebrafish are capable of complete regeneration after removal. Skeletal regeneration and injury repair of zebrafish fins have been studied extensively4,5, while zebrafish scales are a newer bone model in the field but offer advantages for ex vivo culture6.
Scales are highly abundant, with at least 300 scales on each fish that serve as a protective covering for the fish. Each scale is a small mineralized plate consisting of bone-forming osteoblasts and bone-resorbing osteoclasts of a collagen-rich skeletal matrix7. The ossification process of both zebrafish scales and human bones requires the differentiation of mesenchymal stem cells into osteoblasts to form the mineralized matrix. Zebrafish scales offer a great advantage for skeletal research with their strong regenerative ability that can be used to study bone regeneration and repair. However, despite the presence of both osteoblasts and osteoclasts, zebrafish scales lack osteocytes that are important for human bone remodeling and mechanosensation; the superficial location of the scales means that they can be easily removed with a pair of forceps. Upon removal of the scale, a cascade of events occurs, and scale regeneration begins8,9. There are various staining and imaging options available to visualize the activity of osteoblasts and osteoclasts and the mineralization of the scales, as shown in Figure 1. Additionally, the availability of many relevant fluorescent&#160;transgenic reporter lines of zebrafish means that one can visualize cell dynamics during regeneration7,10,11. This process allows one to gain more understanding of de novo&#160;bone formation by observing the early patterning of scale regeneration on the flank of the fish to study morphology, cellular activity, and genetic profiles of these regenerated scales. The biology of scale formation and regeneration has been well characterized. Importantly, scales can show a good predictive ability for therapeutically relevant compounds12 and treatment of fish with glucocorticoids leads to a scale that regenerates to show osteoporotic phenotypes13. The transcriptome of the regenerating scales shows that genes activated in scale regeneration are enriched for those linked to human skeletal diseases, further demonstrating their relevance as a model system6,14.
Finally, these scales can be cultured ex vivo for up to 7 days. Compared to cell line cultures that are typically composed of a single cell type, ex vivo scale culture provides in vitro bone study opportunities within its natural environment containing both osteoblasts and osteoclasts with its natural extracellular matrix8,12,15,16.
Scale culture also allows us to perform drug screening for novel osteoanabolic targets. The abundance of scales on the fish means that one can fill at least two plates of the 96-well plate from just a single fish, allowing for compound screening in a multiwell format where every single well contains one scale along with its natural niche of cells. Additionally, since the scales are thin, drug absorption is predictable12. In summary, the elasmoid scales of zebrafish have great potential in skeletal research and can help us gain more insight into the cellular events during bone formation and repair. Here, we describe protocols for harvesting scales to follow regeneration in vivo and culture the scales ex vivo.

Autor em destaque: Uso de escamas de peixe para pesquisa de remodelação óssea - avanços em imagens ex vivo e interações celulares de dissecação

Regeneração de Escamas de Zebrafish em Toto e Cultura Ex Vivo de Escamas

One of the advantages of using fish scales for research is that they contain both osteoblast and osteoclast, which are essential for bone remodeling. When the scales are cultured ex vivo, we can study the interactions of these two cell populations along with the skeletal matrix, unlike traditional cell culture where there's typically only one type of cell. An advantage of using fish scales for research is that they are abundant on the fish, readily harvested, and they are translucent, allowing better ex vivo imaging.As the scales can completely regenerate, they allow us to study bone regeneration which rarely occurs in mammalian models. The protocols we present here allow us to dissect cell interactions better. This will allow us to test how the different cell types respond to aging and to changes in circadian rhythm.

Watch this Scientific Journal Video about Staining of Harvested Zebrafish Scales at JoVE.com

Coloração de Escamas de Zebrafish Capturadas 

Para realizar a coloração de fosfatase alcalina, comece enxaguando as escamas de Zebrafish colhidas em solução corante de ALP por cinco minutos. Em seguida, coloque as balanças em uma solução de coloração ALP de trabalho por 15 minutos. Em seguida, remova a solução de ALP e lave as escamas em água deionizada para interromper a reação.Para corar as escamas usando o método de coloração de Von Kossa, comece incubando as escamas em solução de nitrato de prata a 5% por 40 minutos sob luz forte. Em seguida, retire a solução de nitrato de prata e lave as escamas em água deionizada por cinco minutos duas vezes. Incubar as escamas em tiossulfato de sódio a 5% durante cinco minutos e, em seguida, lavá-las novamente com água deionizada.Monte as escamas no slide usando um meio de montagem. Cubra-os com uma tampa e visualize-os sob um microscópio. Além do método ALP e Von Kossa aqui demonstrado, existem vários outros métodos de coloração que podem ser usados para visualizar vários aspectos da biologia celular ou da matriz nas escalas, como TRAP, fluorescência do cálcio, vermelho de alizarina e coloração imunohistoquímica.

Research

JoVE Journal

Biology

Staining of Harvested Zebrafish Scales

525 visualizações.  University of Bristol. Para realizar a coloração de fosfatase alcalina, comece enxaguando as escamas de Zebrafish colhidas em solução corante de ALP por cinco minutos. Em seguida, coloque as balanças em uma solução de coloração ALP de trabalho por 15 minutos. Em seguida, remova a solução de ALP e lave as escamas em água deionizada para interromper a reação.Para corar as escamas usando o método de coloração de Von Kossa, comece incubando as escamas em solução de nitrato de prata a 5% por 40...

Vídeo: Coloração de Escamas de Zebrafish Capturadas 

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented.

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new ...