The analysis of activated caspase 3 by wholemount immunofluorescence in zebrafish embryos reveals stage and tissuespecific localization of cells specifically undergoing apoptosis. Fish and dapi staining of the synaptonemal complex of the. Dapi staining of live yeast cells university of texas at austin. Previous studies have shown that the transforming growth factor beta tgf. Stains the nucleus of budding or fission yeast with dapi in order to see it with a fluorescence microscope.
There are several other methods to fix worms previous to dapi staining such as fixation with carnoys solution or formaldehyde that preserve better the integrity of the worm 17. The increase in zebrafish genomic resources together with more sophisticated. Fixationpermeabilization allows good immunofluorescence staining and. Detail protocols for fish can be obtained from a number of practical books but. Following light microscopic analyses, the stained cells were processed for electron microscopy. Prepare metaphase spreads on slides as outlined in a separate protocol. Two dyes, 4,6diamidine2phenylindole dapi and bisbenzimide h33258 have been widely used 68. First, fix and permeabilize cultured cells with a protocol appropriate for your sample. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine.
Counterstain with dapi as the final step in your staining procedure. After the washes, dapi sigma, d9564 staining was performed for 5 min at room temperature final concentration. Harvest cells wash 2x in pbs to get rid of serum proteins. Sample preparation use the fixation protocol appropriate for your sample. To stain nuclei, dilute dapi stock solution with pbs to 0. There may be two reasons why you are not getting anygood dapi staining. Visualizing multiciliated cells in the zebrafish through a combined. The reduced versions, red cmh 2xros and orange cmh 2tmros, are preferred since they are nonfluorescent until entering an actively respiring mitochondrion and oxidized. Dapi 4,6diamidino2phenylindole dihydrochloride is a bluefluorescent dye that binds to atrich regions of doublestranded dna.
In transgenic seca5yfpexpressing fish after 48 hpf not many labeled apoptotic cells could be observed throughout the trunk except several large. Binding is accompanied by an 20fold enhancement in fluorescence, which is directly proportional to the amount of dna present and has an emission maximum at 454 nm. Dapi protocol for fluorescence imaging thermo fisher. After 24 h, transverse sections of the embryos were stained with dapi to.
About frontiers institutional membership books news frontiers social. It is also appropriate for fixation prior to immunostaining, although some antibodies, cell types, target proteins, etc. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. To visualize dna, incubate fixed samples with 100 ngml 4. Would it be possible to stain the embryo with something like hoechst or dapi. Use of dapi as a dna stain for flow cytometry was also demonstrated around this time. Dapi staining is one of the simplest staining and mostly works straight away.
Dilute dapi stock solution to a concentration between 10. Fluorescence increases approximately 20fold when dapi is bound to doublestranded dna. Dapi staining is usually used in cell death detection. Dapi protocol for fluorescence imaging thermo fisher scientific us. Analysis of apoptosis in zebrafish embryos by wholemount. The protocol is improved and robust for optimal sample fixation, tissue clearing and multiprotein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. Cell assay, fix and stain dapi, phalloidin drsctrip. Cryopreservation and in vitro fertilization ivf protocols.
The rationale behind this assay is that mycoplasmafree cultures exhibit only nuclear fluorescence. Simple method for steady, lowlevel embryo production. The whole procedure from embryo preparation, embedding. Labeling fixed cells first, fix and permeabilize cultured cells with a protocol appropriate for your sample.
The concentration of dapi needed for live cell staining is generally very high. Dapi staining is normally performed after all other staining. This material is from the 4th edition of the zebrafish book. If using modified boyden chambers with cells on filters, use dapi below instead of. Protocol to count cell number of preimplantation embryos using nuclear staining with hoechst 33342 or dapi f. Moreover, this method allows potential for imaging gfpreporter lines and. All secondary antibodies were used at a working concentration of 1. I did not find any specific information addressing this problem in my old protocols. One for alla highly efficient and versatile method for. Its use for detecting dna in plant, metazoa and bacteria cells and virus particles was demonstrated in the late 1970s, and quantitative staining of dna inside cells was demonstrated in 1977. To determine the amount needed for your experiment, please note that one tube of solution 1 will yield a total of 100ml of working solution, at a final concentration of 0.
Doing the edu reaction before antibody staining can interfere with staining for. Immunofluorescence protocol with methanol fixation if methanolfixed important. The best resource is probably the protocols you can find in the zebrafish book at. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Here we describe a protocol for wholemount immunolocalization of proteins which is applicable to a wide range of plant species. To facilitate the required incubation steps, whole tissues must be cut into ultra thin 510 mm sections or cut into smaller pieces for whole mount ihc. Add sufficient 300 nm dapi stain solution to cover the cells. Zebrafish neuromast labeling protocols 12205 pattern that allows ready distinction from neuromast cells. Apr 07, 2010 many rb neurons are eliminated by apoptosis within 72 hpf. Pdf embedding, serial sectioning and staining of zebrafish.
Apply 20 l of dapi solution vysis product to the target area and put on cover glass make sure it covers. For icc experiments, cells must be attached to a microscope slide. It is the protocol that i used dapi staining in paraffin. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Dna staining employing fluorescent dyes that bind specifically to dna is the most popular method. In most cases the cells will be spun down and the supernatant removed before adding dapi.
Im working on a protocol using dapi to quickly stain freshly excised tissue. Can anyone suggests the working concentration of dapi for fluorescence studies. Add dapi 1mgml stock to the final concentration of 2. Fluorescence microscopy for cilia in cultured cells and zebrafish. Dapi protocol for fluorescence imaging thermo fisher scientific cn. Dapi 4, 6diamido 2phenylindole is a commonly used dye for bacterial enumeration, or cell counting. Dapi as well as images with specific fish signals can be merged with fish software. Protocol to count cell number of preimplantation embryos. The protocol described here represents a fast way of dyeing worms with dapi, however because of the dissecation of the worm some structures may present some alteration. Note that fixation and permeabilization of the sample are not necessary for counterstaining with dapi. The zebrafish danio reriotumor xenograft model represents. Please refer to the applications section on the front page of the datasheet to determine if this product is validated and approved for use with this protocol. In general you would want the cells at a concentration of 2 x 10 6 ml of dapi. Its blue fluorescence stands out in vivid contrast to green, yellow or red fluorescent probes of other structures.
In another example, the optimal protocol for staining a low abundance protein in a methanol fixed, frozen liver section may require blocking of endogenous biotin and a signal amplification technique. Preparation of zebrafish embryo samples for western blots. Stop the reaction by removing the staining solution and washing the embryos in. Rinse samples once in pbs and then prepare for imaging. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in jb4 plastic resina glycol. Is there a way to livestain the nuclei in the early zebrafish embryo. This is the drscs basic protocol for fixation with paraformaldehyde and staining with dapi andor phalloidin. Cells must be permeabilized andor fixed for dapi to enter the cell and to bind dna. The zebrafish danio rerio has ascended from a predominantly. For now i am testing on formalin fixed tissue, but am seeing a lot of nonspecific dapi staining in some extracellular areas of the tissue. Pellet cells at approximately 2,000 rpm for 5 mins. For example, 5 min is long enough for dissected gonad arms but longer incubation 2030 min is required for whole mount animals.
Prepare needed volume 1mlembryo of dapi dilactate to final working dilution concentration of 10nm in pbs diluted from stock dapi dilactate solution of 10 mm in h 2 o. Dapi nucleic acid stain 3 experimental protocols counterstaining adherent cells for fluorescence microscopy sample preparation use the fixation protocol appropriate for your sample. Fluorecence microcopy, cilia, zebrafish, cultured cells. In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Dapi staining solution ab228549 is a fluorescent stain for labeling dna in fluorescence microscopy. Rather, edu staining uses a rapid clickchemistry reaction between an azide part of staining solution and an alkyne bound to edu, allowing cryosectioned tissue to be labeled at room temperature in dapi staining. Dapi has poor solubility in water, so sonicate as necessary to dissolve.
Labeling fixed cells first, fix and permeabilize cultured cells with a protocol. Add 2 ml of deionized water dih2o or dimethylformamide dmf to the entire contents of the dapi vial to make a 14. However, if fixation is necessary for the downstream experiment or if the formaldehyde fixation is a standard protocol to prepare samples, it can be done in between ctc staining and dapi staining. Fix pelleted cells in icecold 70% ethanol by adding with a pasteur pipette on a vortex. For both applications, following secondary detection. Fish and dapi staining of the synaptonemal complex of the nile tilapia oreochromis niloticus allow orientation of the unpaired region of bivalent 1 observed during early pachytene k.
Mammalian tumor xenografts induce neovascularization in. The dye must be disposed of safely and in accordance with applicable local regulations. In order to obtain the best results, optimization of staining conditions, such as changing the reagent concentration and staining time will be required. In order to create a new protocol or add a comment, you must have a wiki account. There have been suggestions that at higher salinity more than a few % dapi reacts unspecifically with marine bacteria, or binds less effectively to dna zweifel and hagstrom, 1995. This protocol describes the use of dapi to label nuclear dna of cells grown in culture. The 5th edition is available in print and within the zfin protocol wiki.
The staining time depends on the type of sample and varies from 530 min. Stain embryos in dapi pbs solution at room temp, with gentle rocking for 20 minutes. Can anyone suggests the working concentration of dapi for. Any water stage is a good stopping point if you had to do something part way through the procedure. Dapi staining dapi is used to stain dna and in our group is normally used to determine cell cycle information. Dapi is a popular nuclear counterstain for use in multicolor fluorescent techniques. I would like to image the nuclei of the zebrafish embryo for the first 56 hours of. Dapi stains nuclei specifically, with little or no cytoplasmic labeling. For example, 5 min is long enough for dissected gonad arms but longer incubation 20. Store slides covered with sealwrap at room temperature. A simpletouse fluorescent stain, 4,6diamidino2phenylindole dapi, visualizes nuclear dna in both living and fixed cells. Dapi is a known mutagen and should be handled with care. Place flies on apple juice plates and let them lay eggs for 23 days to adjust. Confocal microscopy list fast dapi staining protocol.
I havent been able to find a protocol for passive introduction of a nucleidye. After staining with dapi, detectwith fluorescence microscope or flow cytometry. Visualizing multiciliated cells in the zebrafish through a. Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. Since dapi passes through an intact cell membrane, it can be used to stain live cells and fixed cells. It is labeled nontoxic in its msds and though it was not shown to have mutagenicity to e.
In the protocol described here, colabeling is limited by the requirement for the specific rabbitderived antibody used in our assay. Cell staining reagents the following is a general protocol for preparing assay solutions. The following protocol uses zebrafish adults maintained and cared. Methods for the analysis of early oogenesis in zebrafish. Dapi is a fluorescent dye that stains nucleic acids and is relatively unreactive with inert, nonbiological matter, thus making it useful in differentiating between biotic and abiotic components in a sample. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Collect embryos using paint brush and place them in wash solution 7g nacl, 3 ml tritonx, 997 ml water 3. Insofar as the dab wont autofluoresce or meddle with the dapi fluorescence, at that point it ought to be ok.
I believe that methanol washes may be a way to remove the staining in the yolk, but another zebrafish expert may have more information than me. Why does dapi not stain a paraffin embedded tissue. We have not identified a commercially available nonrabbitderived antiactivated caspase 3 antibody that specifically labels apoptotic cells by wholemount immunofluorescence in zebrafish embryos. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.
If protocol with methanol fixation if methanolfixed cst. Aug 31, 2014 for now i am testing on formalin fixed tissue, but am seeing a lot of nonspecific dapi staining in some extracellular areas of the tissue. Dapi staining was used to determine the number of nuclei and to assess gross cell morphology. Staining nuclei with hoechst or dapi hoechst 33258 bisbenzimide h 33258 exem346460 nm. My protocol is basically the recommended dapi protocol from the manufacturer.
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