Working solutions of 20 mg l pbcl 2, 79 mg l as 2 o 3, and 15 mg l cdcl 2 in embryo medium were prepared in the same way from 5, 10, and 4 g l stock solutions in fish water, respectively. Anyone familiar with an antibody to stain microglia in zebrafish. Both are stained with the antilplastin antibody magenta. Dsred are red and macrophages l plastin positive cells are green. We used regulatory regions of the zebrafish lysozyme c lysc gene to drive. Inflammation drives wound hyperpigmentation in zebrafish by. The plastin also know, as fimbrin protein family belongs to actinbundling proteins, and the protein family is evolutionarily conserved and expressed in yeast, plant. A schematic of the procedure for transient induction of v12rasvsrc in embryos that also have. Novel insights into the conservation of haematopoietic lineages and improvements in our capacity to identify, isolate, and culture such haematopoietic cells continue to enhance our ability to use this simple organism to.
Exosomal release of lplastin by breast cancer cells. Clearing and staining for larval fish cartilage and bone. Lcp1 lplastin antibody aa8172 lsc344326 lifespan biosciences catalog. A variety of different glofish are currently on the market. Plastin 2 is a protein that in humans is encoded by the lcp1 gene. Lcp1 l plastin elisa kit lsf6075 is a 96well enzymelinked immunosorbent assay for the quantitative detection of human lcp1 l plastin in cell lysates, tissue homogenates. I am familiar with 4c4 and lplastin antibodies, but these are not perfect for me. Rabbit antibodies against mouse or zebrafish lplastin, zebrafish pu. Characterization of the human lplastin gene promoter in.
Dynamic changes in microglial and macrophage characteristics. To understand mechanistically the effect of nfil3 knockdown, the expression of hematopoietic cell markers and other genes involved in hematopoiesis was investigated. L plastin antibody detects l plastin protein at cytoplasm and focal adhesion site by immunofluorescent analysis. Lplastin antibody b9 scbt santa cruz biotechnology. Tested in western blot wb and immunohistochemistry paraffin ihc p applications. The actinbinding protein, l plastin, is present in human peripheral blood leukocytes, predominately in monocytesmacrophages, but also in b lymphocytes, t lymphocytes, granulocytes, and natural killer cells. Myelopoiesis in the zebrafish, danio rerio blood american. Methyl mercury suppresses the formation of the tail. By adding a natural fluorescence gene to the fish, scientists are able to quickly and easily determine wh. Model for the study of inflammation and the innate.
The zebrafish as a model organism to study development of the immune system. The hybridomas are stored at the monoclonal antibody. Plastin 1 otherwise known as fimbrin is a third distinct plastin isoform which is specifically expressed at high levels in the small intestine. These macrophages can be distinguished from neutrophils by immunolabeling of l plastin without mpo coexpression and by an elongated morphology. Prophylactic administration of chicken cathelicidin2. Overall, our novel mutant lines establish a sound genetic model and an. For our primary antibody, we used an established rabbit polyclonal. Jan 17, 2018 clearing and staining for larval fish cartilage and bone. Due to their striking genomic similarity to humans, regenerative abilities, and shortened life cycle, zebrafish have become an ideal tool for studying developmental processes, gene expression, and underlying mechanisms of cancer and other diseases. Gene expression and functional analysis of zebrafish larval.
Modeling tuberculous meningitis in zebrafish using. Myelopoiesis and myeloid leukaemogenesis in the zebrafish. Select appropriate zebrafish protein antibodies for your research by isotype, epitope, applications and species reactivity. Zebrafish, inflammation, macrophage, neutrophil, phagocytosis. The glofish is a patented and trademarked brand of genetically engineered fluorescent fish.
Pdf regulation of the cytoskeleton is essential for cell migration in health and disease. This study adds l plastin to a growing list of proteins implicated in t lymphocyte polarity and migration. How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. Lessons from transgenic zebrafish expressing the green. I l compared with control fish i,j, amd3100treated fish do. Zebrafish were the first glofish available in pet stores, and are now sold in bright red, green, orangeyellow, blue, pink, and purple fluorescent colors. Characterization of zebrafish larval inflammatory macrophages. Chapter 9 molecular methods western blots of zebrafish embryos source.
Lplastin antibody pa544200 thermo fisher scientific. Red zebra danio glo fish glow in the dark fwf danios zebra red fish size. Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo. Please use this form to recommend updates to the information in zfin. Fluorescent zebra fish were specially bred to help detect environmental pollutants. The monoclonal antibodies were generated with balbc mice against adult zebrafish protein, and clones were selected based on the specific staining patterns obtained with particular clones. It gets phosphorylated on the serine residue in its headpiece region in response to a variety of inflammatory stimuli. The gene expression was confirmed by using 79 mutant.
It has two actinbinding domains and two ef hand type calciumbinding domains. Targeted deletion of the zebrafish actinbundling protein l. Zebrafish protein antibody scbt santa cruz biotechnology. The expressions of mpx top row, lplastin middle row and mmp9 bottom row were detected in wild type larvae and clo mutants at 6 hpa. Anti l plastin antibody products anti l plastin antibodies can be readily obtained from commercial sources. Aug 01, 2001 the actinbinding protein, l plastin, is present in human peripheral blood leukocytes, predominately in monocytesmacrophages, but also in b lymphocytes, t lymphocytes, granulocytes, and natural killer cells. The l plastin protein is a reported synonym for the human gene lcp1, encoding lymphocyte cytosolic protein 1. Mutant embryos display a general systemic hypoxic response, including the upregulation of hypoxiainduced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response.
Oct 12, 2019 a single domain antibody directed against functional and structural modules of l plastin reduced the association between lfa1 and l plastin, impaired mtoc docking, immune synapse formation and t cell activation. View detailed zebrafish protein antibody specifications by linking to the specific product blocks. It is a sandwich assay which can detect lcp1 l plastin down to 0. As the leading manufacturer of zebrafish antibodies, genetex offers an extensive portfolio of reagents validated for use in this model system. Eurogentec, belgium was purified by affinity matrix and used at 1. Regeneration associated transcriptional signature of retinal. Immunohistochemistry formalinpfafixed paraffinembedded sections anti plastin l antibody ab210099 immunohistochemical analysis of paraffin embedded zebrafish tissue, labeling plastin l using ab210099 at 0 dilution. Select zebrafish protein antibodies from monoclonal antibodies listed below. Identification of regulatory elements recapitulating early. No significant decrease was observed for mmp9 expression arrows, whereas the expression of mpx and lplastin was undetectable n 8, respectively. Here, we extend previous work on this genes mrna localization and show that l plastin is a highly specific marker for the evl and its derivative, the embryonic periderm. Targeted deletion of the zebrafish actinbundling protein. We observed in whole tissues from zebrafish morphants 4 dpf that the expression of pu. The zebrafish as a model organism to study development of the.
L plastin is a leukocyte specific actin bundling protein from fimbrin family of proteins. Antibodies, primary antibodies, zfish zebrafish, alphabetical listing of zebrafish antibodies. Anatomical structures recognized use terms from the zfin. Immunohistochemistry formalinpfafixed paraffinembedded sections anti plastin l antibody ab210099 immunohistochemical analysis of paraffin embedded zebrafish tissue, labeling plastin l. A novel study system for vertebrate tissue growth and repair. The role of nfil3 in zebrafish hematopoiesis sciencedirect. Lymphocyte cytosolic protein 1 lcp1, also called l plastin. Rabbit antibodies to zebrafish mpo and lplastin have been. Immunofluorescent colabeling of zebrafish mpo and lplastin lp revealed two populations of leukocytes that express lp within the caudal hematopoietic tissue cht at 23 dpf.
B, d, f, and h confocal zstack images of l plastin immunofluorescence staining applied on the same embryos to visualize the macrophage population. Over the past ten years, studies using the zebrafish model have contributed to our understanding of vertebrate haematopoiesis, myelopoiesis, and myeloid leukaemogenesis. Ac gastrulation and early segmentation period, lateral view. The membranes were blocked in 5% milk for 1 hour at room temperature followed by overnight incubation at 4c with primary antibodies. Plastin 3 proteins plastins are a family of actinbinding proteins that are conserved throughout eukaryote evolution and expressed in most tissues of higher eukaryotes. For our primary antibody, we used an established rabbit polyclonal designed against the first 111 amino acids of zebrafish l plastin 4649, see experiments in 50. A role for the transcription intermediary factor 2 in zebrafish myelopoiesis. The zebrafish danio rerio has been extensively used in biomedical research as a model to study vertebrate development and hematopoiesis and recently, it has been adopted into varied fields including immunology. About these antibodies all antibodies listed on this page were generated by researchers at the institute of neuroscience, university of oregon liu et al. Remove embryos from their chorions in batches of 100 by placing in 1 mgml of pronase and swirling occasionally 510 minutes for 24 h embryos, 1020 minutes for 3 day embryos. We visualized microgliaimmune cells in control and ouabainlesioned retinas using an antibody to zebrafish l plastin, which is used to mark leukocytes, including. Whereas microglia cells were reduced in number in the entire fish, neutrophils were still present in the ventral trunk area.
L plastin is a leukocyte specific antibody, and tags both macrophages and neutrophils at 48 hpf le guyader et al. By the time this book is published, the availability of the zebra. Egfpkj2 line, to pamela raymond for sharing the opn1sw1, and rhodopsin in situ probes and michael redd for sharing the l plastin antibody. Jan 26, 2012 we are very grateful to kenneth poss for the gift of the dnfgfr1 transgenic fish line, which enabled this study in the first place, to shoji kawamura for sharing the tg5. Visit the antibody supplier page for key details, including validated applications and target specificity. Is anyone familair with, for example, a p2y12 ab that works in zebrafish. It contains a potential calciumbinding site near the n terminus. Investigating microgliabrain tumor cell interactions in.
D immunostaining of innate immune cells with anti l plastin antibody shows the presence of leukocytes around the wound in control fish. This antibody reacts with bovine, canine, equine, guinea pig, human, mouse, rabbit, rat, zebrafish samples. Here we characterize the morphology and function of inflammatory macrophages in zebrafish larvae. Origins and unconventional behavior of neutrophils in developing. The cterminal 570 amino acids of the t plastin and l plastin proteins are 83% identical. The fulllength protein is 70,288 daltons in mass with 2 identified isoforms.
Phagocytosis of mycobacteria by zebrafish macrophages is. Zf l cells were derived from normal adult zebrafish liver, and have been growing in culture for more than 100 generations. Lplastin is a leukocytespecific protein that crosslinks actin filaments into tight bundles, increasing the stability of actinbased structures such as podosomes and lamellipodia. Zebrafish have emerged as a powerful model system to study leukocyte recruitment and inflammation.
The assay consists of microinjecting a single cell suspension of fluorescently labelled m. Pdf targeted deletion of the zebrafish actinbundling. We appreciate as much detail as possible and references as appropriate. The cells were derived in basal nutrient medium supplemented with fetal bovine serum fbs, trout serum, trout embryo extract, bovine insulin and mouse epidermal growth factor. Sophisticated regulation of the actin cytoskeleton by a variety of actinbinding proteins is essential for eukaryotic cells to perform their diverse functions. Lplastin antibody lsc344326 is an unconjugated rabbit polyclonal antibody to zebrafish lplastin lcp1. Due to their striking genomic similarity to humans, regenerative. Regeneration associated transcriptional signature of. Fgf signaling is required for photoreceptor maintenance in. The polyclonal antibody against zebrafish flt1 extracellular domain cqvtsgpskretntt. In humans, two ubiquitous plastin isoforms l and t have been identified. Flt1 acts as a negative regulator of tip cell formation.
Plastins are a family of actinbinding proteins that are conserved throughout eukaryote evolution and expressed in most tissues of higher eukaryotes. Genomic dna spanning the predicted promoter region. However, the ability to observe and characterize such interactions, in realtime, within a living vertebrate has proved elusive. Pdf a role for the transcription intermediary factor 2 in. The l isoform is expressed only in hemopoietic cell lineages, while the t isoform has been found in all other normal cells of solid tissues that have replicative potential fibroblasts, endothelial cells, epithelial cells, melanocytes, etc. Although l plastin is also present in neutrophils, the alexa 568 fluorescence signal is not visible in. Identification of a conserved and acute neurodegeneration. For lplastin staining we incubated in alexafluor488 invitrogen a11034, 1. Targeted deletion of the zebrafish actinbundling protein lplastin. For these reasons, we have begun to examine the structure of the l plastin gene with emphasis on the characterization of its promoter and its regulation in normal and transformed cells. Zebrafish antibodies primary antibodies sigmaaldrich.
The zebrafish lysozyme c promoter drives myeloidspecific. Human lcp1 lplastin quant elisa kit sandwich lsbio. Singledomain antibodies are the antigenbinding fragments derived from hcabs showing several beneficial properties e. Derivation and characterization of a zebrafish liver cell. Cellular visualization of macrophage pyroptosis and interleukin1.
Red zebra danio glofishglow in the dark product view. Use, applications and mechanisms of intracellular actions of. The discovery of heavychainonly antibodies hcabs in camelids and sharks led to the rise of a new research field in which singledomain antibodies are used for various applications. An antibody against the zebrafish lymphocyte cytosolic protein 1 l plastin. Alternate splicing results in multiple transcript variants. Zebrafish danio rerio, a minnowlike freshwater fish, has emerged as a popular animal model for research and development. Despite lplastins role in both immunity and cancer, in zebrafish there are no. View our alternatives for ab81454 or you can download the archived datasheet pdf from this page. Gtx124420 ihcwm image lcp1 antibody detects lcp1 protein on whole mount zebrafish by immunohistochemical analysis. The lplastin protein is a reported synonym for the human gene lcp1, encoding lymphocyte cytosolic protein 1. I a similar larvae to that in h, but with vsrcexpressing cells green. I am doing in situ hybrdization on mouse embryos for a novel gene and so have to. The monoclonal antibodies were generated with balbc mice against adult zebrafish protein, and clones were selected based on the.
1268 310 920 1257 1411 141 685 323 969 1031 1200 990 347 637 761 818 978 911 987 1085 1103 346 648 306 744 1340 557 909 982 1157 322 622 845 1481