Capuco 3 department of animal and avian sciences, university of maryland, college park 20742. Current methods to visualize mineralized tissue in zebrafish rely on. Acridine orange staining and visualization in zebrafish to. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. We analyzed postembryonic neurogenesis in zebrafish brain using the thymidine analogue. Immunolabeling was performed as described in the zebrafish book. Plate cells in 96well plate and incubate with respective test substance. Bromodeoxyuridine brdu labeling and immunohistochemical. Invert tube until the solutions are clear 3 inject mouse intraperitoneal with 10 lgram of body weight of brdu solution 10 mgml. Brdu staining protocol co staining with anti brdu brdu can be used in conjunction with other antibodies to identify proliferating and newly differentiated neurons. C close up of head in ventral view of alcian blue cartilage staining at 3dpf. Dna staining protocol for flow cytometry for research use only.
Bromodeoxyuridine brdu is a thymidine analog and it incorporates into the dna only if the cells are at sphase. Any water stage is a good stopping point if you had to do something part way through the procedure. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. A simple wholemount staining protocol for bone and or cartilage in adults and larvae clearing and staining for larval fish cartilage and bone chromosomes spreads.
Hernandez and coworkers used brdu labeling and transgenic gfp reporter lines. Analysis of apoptosis in zebrafish embryos by wholemount. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. The entire staining procedure requires approximately 3 hours. If i follow exactly the roche provided protocol i get 100% brdu positive cells and cytosolic staining with a 3h brdu pulse. This feature can be used to trace the cell lineage and cell sur vi al. Postembryonic neurogenesis in zebrafish danio rerio brain. Morton nov 2010 brdu incorporation and detection in vivo protocol 022410 method a. Fixation of the sample containing the target cells. Preparation of zebrafish embryo samples for western blots.
Flowfish fluorescent in situ hybridization is a cytogenetic technique to quantify the copy number of specific repetitive elements in genomic dna of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols. Brdu kit for ihcicc immunofluorescence materials provided brdu fixation buffer. Incubate cells in culture with 10 m m brdu sigma for an appropriate time 30 mins hour plus. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. Analysis of cell proliferation, senescence and cell death in. Timeline of drug treatment and method of quantification of cell number. I know roche offers a kit that has anti brdu antibody with nucleases no acid or heat needed but the kit comes with enough brdu labelling to label a city but only enough antibody for a few slides and it cant be savedreused. Brdu cell proliferation protocol cell signaling technology. Feel free to add new protocols to the appropriate section or add comments to any existing protocol.
Protocol for fasc analysis of cell cycle using brdu and pi labeling of cells with brdu. A, antibody staining against phosphorylated histone h3 in wildtype 24hpf embryos. Flow cytometry protocol for cell cycle analysis using brdu, an analog of thymidine, readily incorporated into dna during dna synthesis. In the protocol described here, colabeling is limited by the requirement for the specific rabbitderived antibody used in our assay. Cells can be pulselabeled with brdu, and those cells that are synthesizing dna in sphase of the cell cycle will incorporate brdu into.
Cells from all time points were concurrently stained. The zebrafish danio rerio has ascended from a predominantly. I could find a few publications using edu staining in zebrafish embryos, and referring to the invitrogen kit. Valproic acid, a histone deacetylase inhibitor, regulates cell. The utility of ki67 and brdu as proliferati e markers of. At this stage the cartilage staining is diffuse and nonspeci. Unlike many lengthy labeling and clearing protocols for thick tissue, our. Upper and lower panels are dorsal and lateral views, respectively. Ki67 a cellular marker for proliferation, the ki67 protein is present in cells at cycle phases g1, s, g2 and m, but absent in resting g0 cells. At each time point 0, 3, 6, 9, and 24 h after brdu incubation, 5 ml of cells was pipetted into a fresh tube, washed with 1x hbss, fixed using bd cytofixcytoperm buffer, washed, and stored at 4.
For convincing and publishable results, i would use tunel or caspace3 staining for the reasons mentioned above. Remove culture medium from cells and replace with brdu labeling solution. Background nestin is expressed in neural progenitor cells npc of developing brain. After brdu labeling, an additional dna hydrolysis step sometimes referred to as a. Spin down nuclei expect a very small pellet, aspirate supe and vortex pellet. To measure dna synthesis or cell proliferation, 5bromo2deoxyuridine brdu can be incorporated into dna in place of thymindine. Add brdu sigma at a final concentration of 10 um to approximately 1 x 106 cells, and incubate under the appropriate growth conditions for 15 to 60 minutes to pulse label the cells. This technique has been used to label replicating cells in zebrafish embryos larison and bremiller. Ct techniques, that are ultimately limited in resolution. Scientific protocols bromodeoxyuridine brdu labeling. Zebrafish embryology and cartilage staining protocols for. The labeling of cells seems to work fine but the fluorescent signal for the brdu is very weak when i flow the cells.
Brdu is incorporated into replicating dna and can be detected using antibrdu antibodies. Leave cells at 4 o c from 30 mins to a week pellet cells at approximately 2,000 rpm for 5 mins. Not for use in diagnostic or therapeutic procedures. I dont have a definitive answer but i think that the brdu protocol increased background fluorescence, fitc channel in my case. Cycling cells are incubated with 5bromo2 deoxyuridine brdu, a synthetic analog of thymidine which incorporates into newly synthesized genomic dna during the sphase of mitosis. To visualize cell proliferation, we exposed the embryos to brdu for 1 hr and. Simple method for steady, lowlevel embryo production.
Brdu bromodeoxyuridine 5bromo2deoxyuridine is an analog of the nucleoside thymidine used in the brdu assay to identify proliferating cells. Brdu flow kit staining protocol the bd pharmingen brdu flow kit staining procedure offers several options forsample handling. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Cycling cells are incubated with 5bromo2deoxyuridine brdu, a synthetic analog of thymidine which incorporates into newly synthesized genomic dna during. I know roche offers a kit that has antibrdu antibody with nucleases no acid or heat needed but the kit comes with enough brdu labelling to label a city but only enough antibody for a few slides and it cant be savedreused.
Brdu labeling is a reliable method to locate the cells that are actively dividing. I am trying to establish a brdu flow protocol in combintion with dna staining pi for stem cells in our laboratory and i have a couple of problems with that. A protocol on diverse applications of brdu labeling can be found in protocol series by jackson and cook 2 3 4. Immunofluorescence protocol to detect brdu incorporation. Dear erin, we have seen such a shift in ki67 staining in the past when measuring it together with brdu. Further understanding of cell cycle regulation in zebrafish embryos was. In the optic tectum teo, brdu labeling was observed in the superficial, dorsal, and.
B, brdu incorporation to mark cells in s phase in the tail of a 28hpf wildtype embryo, c and d, apoptotic cells can be visualized by tunel c. Clearing and staining for larval fish cartilage and bone. Here we describe the steps required for brdu labeling in adult zebrafish brain starting from brdu injection to the final analysis in the microscope. However, when no marbles are in the tank, the fish will rapidly consume the eggs. It is recommended that as incubations can be very long in whole mount staining, the antibody should be diluted in blocking buffer containing 0.
Anti brdu antibodies can then be used to detect proliferation. Brdu staining protocol introduction brdu labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. Circadian timing of injuryinduced cell proliferation in zebrafish. Fix fish in 4% paraformaldehyde in pbs for 2 x overnight at 4c. Although the species is relatively easy to maintain in the laboratory, there are a number of management. However, brdu is not a marker of the sphase of the cell cycle. Brdu labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. Surface stain with pe and cychromeconjugated antibodies use 1x pbs as buffer, wash 1x pbs cf 610 min 1200 rpm. The ebioscience brdu staining kit for flow cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells by flow cytometric analysis. Fish and marbles at the onset of the light cycle, zebrafish will generally initiate breeding behavior that results in the laying and fertilization of eggs.
The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate. Wash with triton x100 permeabilization buffer 3 times, 2 minutes each add fluorescently labeled secondary antibody. Brdu, and then resuspended in 25 ml of fresh medium. The normal method of brdu if requires heat or acid to denature the dna with seems to be destroying the gfp epitope. Steps include preparation of reagents and methods for staining with rabbit and mouse anti brdu antibodies such as mouse anti brdu antibody, clone bu20a mca2483 and rabbit anti brdu antibody ahp2405.
Bromodeoxyuridine an overview sciencedirect topics. The labeling of cells seems to work fine but the fluorescent signal for the brdu is very weak when i. The brdu staining kit for flow cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells by flow cytometric analysis. Brdu antibody staining protocol for immunohistochemistry. Brdu antibody staining protocols for flow cytometry and immunohistology. Culture cells in appropriate vessel for microscopy. The complete and updated rotifer polyculture method for. Using a brdu incorporation assay with a short labeling period, we. Fix pelleted cells in icecold 70% ethanol by adding with a pasteur pipette on a vortex. Remove this solution and add 1 ml of antibody staining buffer. As a thymidine analog, it is a marker of dna synthesis.
Samples were prepared for brdu detection according to the following protocols. Flowfish is most commonly used to quantify the length of telomeres, which are. Avoid contact with skin, eyes, and mucous membranes. But ive used ao staining for a rough cell death assay in zebrafish and it did. Fixation stabilizes macromolecules and cytoskeletal structures thus preventing lysis of the cells during hybridization. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow. Analysis of cell proliferation, senescence and cell death. For rapidly growing cells a pulse of 2 hours is enough to label a significant fraction of the cells. In contrast, vpa reduced cell proliferation in the adult zebrafish optic tectum. Flowfish is most commonly used to quantify the length of telomeres, which are stretches of repetitious dna.
Steps include preparation of reagents and methods for staining with rabbit and mouse antibrdu antibodies such as mouse antibrdu antibody, clone bu20a mca2483 and. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon older literature referred to zebrafish as brachydanio rerio. Brdu labeling and detection protocol thermo fisher. Brdu incorporation and detection in vivo protocol 022410. In a tank that has been marbled, the eggs collect between the marbles and escape predation. Bromodeoxyuridine brdu, an analog of thymidine derivative of uridine, is a uridine derivative that can be incorporated specifically into dna in place of thymidine. This material is from the 4th edition of the zebrafish book. Brdu staining label thymocytes by continuous administration of brdu sigma b5002 via drinking water at 0. Cell proliferation in the developing lateral line system of zebrafish. Jan 19, 2016 the correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration.
The following protocol is designed to label a fraction of proliferating cells in zebrafish embryos, to allow. Incubate for 1 to 4 days on a gentle rotation devise at 4oc. At the same time fixation permeabilize the cell walls for the fluorescentlylabeled oligonucleotide probe molecules. The 5th edition is available in print and within the zfin protocol wiki. Histological methods protocols zfin community wiki. Dna, unlike larger antibrdu primary antibodies, making it well suited for. A representative image of brdu staining of zebrafish caudal fin under ld conditions at zt 9. Cells can be pulselabeled with brdu, and those cells that are synthesizing dna in sphase of the cell cycle will incorporate brdu into the dna. Cytometry brdu and ki67 staining purdue university. We have not identified a commercially available nonrabbitderived antiactivated caspase 3 antibody that specifically labels apoptotic cells by wholemount immunofluorescence in zebrafish embryos.
The zebrafish danio rerio is a preeminent laboratory animal utilized in a growing number of scientific disciplines, including but not limited to developmental genetics, toxicology, behavior, aquaculture, regenerative biology, and the modeling of many human disorders 15. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Brdu staining reveals cells proliferation at the jaw joint is affected by wnt16. One useful feature of brdu is its longterm retention in divided cells and its passage to their daughter cells. Spins are done in a clinical tabletop centrifuge 5 at top speed, approx x g 1. Brdu incorporation and gfp staining protocol online. A spectacular example of such use was a study by eriksson and colleagues who injected patients with brdu and then examined brain tissue postmortem up to 2 years.
Brdu staining protocol costaining with antibrdu brdu can be used in conjunction with other antibodies to identify proliferating and newly differentiated neurons. With this staining protocol, it is possible to immunofluorescently stain and analyze samples in a single day. Useful techniques for the study of the cell cycle, proliferation or apoptosis as shown in zebrafish embryos. Aliquot and freeze down at 20c until needed 2 thaw brdu at rt. Immunofluorescence protocol to detect brdu incorporation 1 plate cells 24 hours in advance, add brdu companies supply x or 500x solutions and incubate from 2 to 6 hours depending on your experiment. The epub format uses ebook readers, which have several ease of. For simultaneous brduph3 stain, add a fluorescent antirabbit antibody as well. Brdu injection 1 dissolve brdu 10 mgml in sterilized pbs. Dna staining protocol for flow cytometry bd biosciences. We will shoot for 6075 minutes of lecture, followed by questions, so that we can return to the lab by 3. Would someone have a detailed edu labelling protocol for. However, i would be interested in a detailed protocol edu concentration used, type of. Cells, which have incorporated brdu into dna, can be quickly detected using a monoclonal antibody against brdu. Despite its wide use as an npc marker, the function of nestin in embryo development is unclear.