It is used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other. Adjusting the ph of the alcian blue solution allows the demonstration of subtypes of acid mucins. A rapid and nondestructive protocol for wholemount bone. Zebrafish represent a vertebrate model organism that has been widely, and increasingly, employed over the last decade in the study of developmental processes, wound healing, microbehost interactions, and drug screening. Additionally, we focus on skeletal mutant phenotypes in this protocol and include a procedure for alcian blue alizarin red staining modified from previous protocols 15. Clearing and staining for larval fish cartilage and bone. Threespine stickleback are an important model for behaviour and evolutionary studies. Standard protocols for monitoring and sampling zebra mussels. I followed the protocol through but saw no staining. Does anyone have a protocol for cellular staining with coomassie. Pierre massons 18801959 original formulation have different specific applications, but all are suited for distinguishing cells from surrounding connective tissue most recipes produce red keratin and muscle fibers, blue or green collagen and bone, light red or pink cytoplasm, and. To test compatibility of the echofish protocol with other fluorescent labeling techniques, we stained hippocampal cultures with a panel of antibodies map2, gfap, and synaptophysin or with fluorescent phalloidin, followed by echofish. Towards an alternative for the acute fish lc50 test in chemical assessment. Now lets look at the specific steps used for alizarin red, alcian blue, and alizarine yellow dyes.
Ap staining works very well after an antibody stain. Hematoxylin eosin staining in rat brain characterization of an animal model of glioma c6 cells injection in the putamen. A fresh look at zebrafish from the perspective of cancer research. Immunohistochemical staining with alcian blue is a convenient method to determine the extent of sulfation of mucins in tissues.
Last updated february 23, 2008 zebrafish international resource center zirc 5274 university of oregon eugene, or 974035274, usa phone. Analysis of zebrafish larvae skeletal muscle integrity with. Alcian blue cartilage and alizarin red bone publications. Laboratory 6protein isolation from fish and western blotting. Phthalocyanines are synthetic analogues of porphyrins that contain nitrogenaromatic rings surrounding a central atom. Alizarin red s stained specimens lose staining over time. We will shoot for 6075 minutes of lecture, followed by questions, so that we can return to the lab by 3. A quick and simple fish protocol with hybridizationsensitive. The impact of alcian blue staining on danio rerio embryos. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Here we propose a new protocol for wholemount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. Remove the fat from the mice but be careful that damage is not done to the specimen. The laboratory zebrafish laboratory animal pocket reference.
The protocols only describe one or two common methods for each type of data collection. They play roles that are as diverse from regulating gene expression, to playing a role in maintaining the proper cellular structure, sensing the environment, as well. Multiplex fluorescence in situ hybridization mfish. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. A 1 ml volume will be required for each cell sample. The original zebrafish or zebra danio, danio rerio is a native of rivers in india and bangladesh. Calcein, another standard reagent for in vivo skeletal staining of zebrafish, was used as a control staining, following an established calcein staining protocol. Useful tip for better zebrafish wholemount ihc staining the zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Alizarin red protocols the alizarin red dye is first mixed with an acid, such as hydrochloric acid. Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. A growing number of quantitative trait loci qtl and gene expression studies are identifying genes related to ecologically important traits in.
Coomassie brillant blue r250 cbr250 and silver staining are the most widely used methods for the routine visualization of proteins separated by sdspage. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. Common routine stain not an immunohistochemical stain to detect mucins wikipedia. Dilute the dapi stock solution to 3 m in staining buffer 100 mm tris, ph 7. In most protocols, sections are stained with the standard alcian blue ph 2. Glucocorticoids alter craniofacial development and. This protocol describes the standard cbr250 staining method, along with a simple method for preparing stained gels for longterm storage. Upper and lower panels are dorsal and lateral views, respectively. Can you provide a protocol to stain the zebrafish with oil.
Check clarity of solution under a microscope to make sure there are no precipitates. Staining procedure deparaffinize sections and hydrate to running tap water using the automatic stainer. Dissect mice by removing the skin and organs completely. Cbr250 is an organic dye that complexes with basic amino acids, such as arginine, lysine, and histidine, as well as tyrosine.
Zebrafish embryology and cartilage staining protocols for. A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press. Although you can stain both cartilage and bone in the same specimen, you need to be aware that bone staining can mask cartilage staining, for instance when perichondral ossification takes place around a cartilage template. In this experiment, zebrafish 8 or 9 day post fertilization must be stained with oil red o staining to visualize the lipids. The chemical protocol was inspired by fleming and collaborators experiments 63. Embryos to be used for alcian blue staining were grown in 0. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Negative stain negative staining is an excellent way to determine an organisms cellular morphology. It should, however, be noted that ars concentrations used here were much lower 0. A 9 dpf zebrafish control larvae head skeleton presenting calcified calcified pharyngeal teeth cptwhile other structures like meckels cartilage mc and ceratohyal ch remain as cartilage.
Saucedo and andrea james school of biological sciences university of northern colorado, greeley, co contact. Zebra is a privately held preclinical stage biopharmaceutical company. However, when no marbles are in the tank, the fish will rapidly consume the eggs. A fluorescence in situ hybridization fish protocol for. Zebrafish bone and general physiology are differently affected by. Staining and observation of cartilaginous structures in. Wash in saturated sodium borate solution for 912 hours. Characterization of zebrafish larval inflammatory macrophages. Assay procedure equilibrate all materials and prepared reagents to room temperature just prior to use and gently agitate. Stegostomatidae contains the zebra shark stegostoma fasciatum, and rhincodontidae contains the whale shark rhincodon typus. Staining protein gels protocols thermo fisher scientific uk. Alcian blue ab is a cationic dye derivative from the group of phthalocyanines. Visualization of skeletons and intervertebral disks in.
Stain in alcian blue solution for 20 seconds in the microwave. Additionally, this protocol provides guidelines on how. Thus, optimization of the staining condition was required before this experiment. Loizides, georgiou, somarakis, witten, and koumoundouros 2014 used the walker and kimmel 2007 protocol followed by glycol methacrylate embedding for histological analysis. The critical electrolyte concentration principle was applied to the alcian blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. Multiplex in situ hybridization m fish is a 24color karyotyping technique and is the method of choice for studying complex interchromosomal rearrangements. Haematoxylin and eosin stain project gutenberg self. Stains for mucin alcian blue pas mayer mucicarmine hale colloidal iron stain 16. Special stain in histopathology linkedin slideshare. Observe the slide under the microscope, using proper microscope technique. We report double mutants disrupting two zebrafish kinesini genes hereafter kif5blof that we. Add colcemid for last 4 hrs at 200 ngml 10,000x of 2 mgml stock, ie 1. Alcian blue staining protocol zebrafish best picture of.
Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. The staining of the motor neurons and the somites in the developing zebrafish is a difficult task. Alcian blue staining was performed using an acidfree protocol 87. Alcian blue stains acid mucosubstances and acetic mucins. Alcian blue principle alcian blue is a copper phthalcyanin dye and contains positively charged groups capable of salt linkage with certain polyanions. The oxidation product of haematoxylin is haematin, and is the active ingredient in the staining. Repeated blood collection for blood tests in adult zebrafish. In the following example, serial sections of the colon from wt and glcnac6st2deficient ko. Alcian blue alizarin red skeletal staining october 2003 eddy m. Staining is complete when the gel is no longer visible in the dye solution. The company is pioneering the development of platform technologies that allow cellular functionbased screening for the discovery and selection of fully human therapeutic biologics antibodies, peptides or proteins from dnaencoded combinatorial libraries of human antibodies, peptides or proteininproteins. Histologystaining protocols your labs reference book.
This protocol covers staining of the pre and postnatal mouse skeleton using alcian blue and alizarin red to identify cartilage and bone, respectively. A twocolor acidfree cartilage and bone stain for zebrafish larvae. 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. It is the most widely used stain in medical diagnosis. Cells isolated by facs were plated on glass slides and stained using leukocyte peroxidase mpo activity kit sigma according to manufacturer protocols or cells were affixed.
With the increase in the laboratory use of zebrafish, several advantages, such as a high genetic homology to humans and transparent embryos, which allow clear disease. More blue staining new collagen fibrils was observed in sfnpvegf implant at 4 and 8 week. Place in 2nd eosin and attach to automatic stainer. Dapi counterstaining protocols thermo fisher scientific jp. Skeletal development in zebrafish using alcian blueali. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde pfa at 4c overnight. Flow fish is most commonly used to quantify the length of. A simple wholemount staining protocol for bone and or.
Coomassie brilliant blue g250 differs from coomassie brilliant blue r250 by the addition of two methyl groups. Researchers have developed a new, more efficient way of cloning zebra fish, a breakthrough. Laboratory 7protein isolation from fish and western blotting proteins are the most important macromolecule in the cell, as they are responsible for almost all cellular functions. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle. They are pas negative but do stain selectively with alcian blue at ph 0. Staining and observation of cartilaginous structures in zebrafish complete larvae introduction. Alcian blue staining of gags in tissue may be modified through the addition of an electrolyte such as magnesium chloride. I saw in short, two kind of staining with alcian blue in acetic acid ph 2.
Massons trichrome is a threecolour staining protocol used in histology. C close up of head in ventral view of alcian blue cartilage staining at 3dpf. The new procedure that is rapid and nondestructive and allowed to obtain highly cleared specimens was based on our rap system for bone staining of whole mount. At this stage the cartilage staining is diffuse and nonspeci. To save time in largescale experiments, a doubleteam approach is recommended to perform blood collection. A rapid and nondestructive protocol for wholemount bone staining.
Alcian blue staining of cartilage for electron microscopy. In a tank that has been marbled, the eggs collect between the marbles and escape predation. Therefore, we include a kasp protocol in this work. Imaging blood vessels in the zebrafish sciencedirect. Bc 9 dpf control zebrafish head b and trunk c presenting no signals of bone calcification. May 12, 2015 to date, several protocols have been developed to achieve clear and identifiable metaphase chromosome spreads from larvae of a single fish species. Empirical formula hill notation c 56 h 68 cl 4 cun 16 s 4. Dreissenid zebra and quagga mussel monitoring protocol annually the dnr staff selects over 100 water bodies to sample for zebra and quagga mussel infestations.
Zhiyuan gong and his colleagues at the national university of singapore were working with a gene that encodes the green fluorescent protein gfp, originally extracted from a jellyfish, that naturally produced. A collection of western plot protocols for research, provided by invitrogen. The combination of the alcian blue and the pas techniques can be used as a means of distinguishing neutral mucins from acid mucins. The arrows indicate positive staining of new collagen fibrils. Whole mount sudan black staining of neutrophils was performed as described except that a 1. Braunbeck t, bottcher m, hollert h, kosmehl, t, lammer e, leist e, rudolf m, seitz n. During this incubation time, examples of other vertebrate stainings using alcian blue were passed around, such as a mouse embryo. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics. This work investigated the dependency of chromosome preparation parameters including colchicine concentration 0. Larvae subjected to the intestinal inflammation treatments were fixed in 4% paraformaldehyde, rinsed in acidic ethanol and stained with alcian blue. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2fold higher than that of proq emerald 488, a fluorescent periodate schiffbase stain. If any one has a working protocol of oil red o staining pertaining to.
The stain molecules carry a positive charge and are attracted to the negative mucins. Any water stage is a good stopping point if you had to do something part way through the procedure. A alcian blue staining of head cartilage representing the landmarks used for. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. However, staining cartilage with alcian blue would affect subsequent bone staining with alizarin red s.
Here we describe a novel staining method, designated succinylation alcian blue staining, for visualizing mucins on a pvdf membrane. Staining bones with alizarin red s after clearing away the soft tissue. Walker and kimmel 2007 proposed an acidfree double staining protocol suitable for early stages of zebrafish. It measures three centimeters long and has gold and dark blue stripes. However, the efficiency of these protocols in more than one fish species has barely been compared within a single study. Alcian blue stain reveals defective chondrocyte stacking insets in kn. Alcian blue has been used to stain hyaluronan ha, a nonsulfated gag, but the intensity of the staining of ha is affected by the method of tissue fixation used. Jul 08, 2011 the echofish protocol is compatible with other fluorescent cellular labeling techniques. Alcian blue staining for embryos or young larvae 1.
Kinesin1 promotes chondrocyte maintenance during skeletal. The distribution and structure of material in glutaraldehydefixed cartilage stained at ph 5. A skillful researcher will take 12 min to carry out the blood collection procedure protocol 2. In this study, we describe a straightforward method to perform evans blue dye ebd analysis on zebrafish larvae. Alcian blue is a basic dye containing copper, which gives it the blue color. A handbook on the nature and uses of the dyes employed in the biological laboratory classic reprint on free shipping on qualified orders. Dreissenid zebra and quagga mussel monitoring protocol. The most commonly used dye for visualizing proteins in sdspage gels is coomassie brilliant blue r250 cbr250 because of its relatively high sensitivity. Teleost fish such as zebrafish danio rerio are increasingly used for. Optimized for 21 dpf zebrafish juveniles but it works well on other similarly. The other families in the order are brachaeluridae, the blind sharks. A 9 dpf zebrafish control larvae head skeleton presenting calcified calcified pharyngeal teeth cptwhile other structures like meckel. The zebrafish is a quickly developing organism, usually resembling the adult zebrafish after one day.
Shifting zebrafish lethal skeletal mutant penetrance by. This protocol is designed to assist dnr staff in collecting samples for adult and veliger larval form dreissenid mussels. To wash the samples, the students removed the stain from the dishes with a small transfer pipette and added acid ethanol. Flow fish fluorescent insitu 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. Zebrafish embryo acute toxicity test to predict short term. Skeletal development in zebrafish using alcian blue alizarin red double staining. Excessive amounts of nonsulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis.
Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. Useful tip for better zebrafish wholemount ihc staining. Standard staining procedures the internet pathology laboratory for medical education, florida state university college of medicine standardized staining procedures for histology laboratory. A coppercontaining dye used as a gelling agent for lubricants, for staining of bacteria and for the dyeing of histiocytes and fibroblasts in vivo.
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