In the present study, we applied coomassie brilliant blue staining to. Alcian blue staining protocol by roy ellis ihc world, llc. 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. Using this method, we identified docking protein 2 dok2 as. Coomassie dye recipe the order of preparation is critical. Invitrogens pcna staining kit uses a biotinylated pcna monoclonal antibody clone pc10, thus eliminating the need for a speciesspecific secondary antibody. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Be sure to note in your notebook, which fish sample is in each lane. Analysis of apoptosis in zebrafish embryos by wholemount. Principle this is a combined method utilising the properties of both the pas see pas and alcian blue see alcian blue methods to demonstrate the full complement of tissue proteoglycans. Understanding on the mechanisms of vascular branching morphogenesis has become a subject of enormous scientific and clinical interest. Replace the staining solution with destaining solution i. Recently, pena1980 succeeded in staining cytoskeletal systems of cultured fibroblasts at a level of light microscopy with coomassie brilliant blue r250 which is widely used for staining protein in electrophoresis.
Histologystaining protocols protocol online your labs. Coomassie blue r250 permanently stains membranebound proteins and is compatible with pvdf and nitrocellulose membranes, but it is incompatible with nylon membranes. We strongly recommend that you mix your own coomassie stains see protocol below for the recipe. Gel may be prefixed in 50% meoh, 10% hoac, 40% h 2 o for 30 minutes to overnight. Coomassie blue staining thermo fisher scientific sg. Protein concentrations of solubilized determined with bradford method 27. Connect the leads to a power supply red to red and black to black, and electrophorese the samples until the bromophenol blue dye front has traveled to the. Add colcemid for last 4 hrs at 200 ngml 10,000x of. Acidfree protocols were adapted 65 to perform alcian blue 8 gx sigmaaldrich, diegem, belgium staining of cartilage. About 100200 embryos are readily available every week from a single pair of adult fish. Wadeable stream fish community protocol page 61 chapter 6. Lillies sulfuric nile blue method for melanin and lipofuscin. A simple wholemount staining protocol for bone and or. Invitrogen pcna staining kit thermo fisher scientific.
We have not identified a commercially available nonrabbitderived antiactivated caspase 3 antibody that specifically labels apoptotic cells by wholemount immunofluorescence in zebrafish embryos. Alcian blue pas stain protocol histopathology service. Recommended sds page stain protocols kits like gelcode blue from pierce and biosafe coomassie from biorad are not compatible for ingel digestion and mass spectrometry analysis unless you do a fixing step first. Improved coomassie blue dyebased fast staining protocol for. Here is the protocol for alcian blue periodic acid schiff ab pas. Biopals prussian blue cell staining reagent pack has been formulated for use with molday ion and other iron based nanoparticles. Revisiting in vivo staining with alizarin red s a valuable approach. Click the section headings blue to expandcollapse material. The time required to visualize proteins using coomassie blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a coomassie blue dye solution at boiling temperatures. Add colcemid for last 4 hrs at 200 ngml 10,000x of 2 mgml stock, ie 1. Immunostaining of dissected zebrafish embryonic heart. Application of coomassie brilliant blue staining to cultured.
I followed the protocol through but saw no staining. Staining is complete when the gel is no longer visible in the dye solution. Mar 08, 2018 intestine of ballan wrasse labrus bergylta a species of marine fish was stained with alcian blue pas at ph2. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. Shop online for a wide selection of fisher healthcare protocol gram stain sets reusable tray provides secure bottle storage. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Remove destaining solution i and replace with destaining solution ii. Zebrafish, which have small, accessible, transparent embryos and larvae, provides a unique living animal model to facilitating highresolution imaging on ubiquitous and deep localization of vessels within embryo development and also in adult tissues. Alcian blue ab is a cationic dye derivative from the group of phthalocyanines. Staining gels with coomassie blue r250 or coomassie blue g250.
As a result, pcna staining is exceptionally clean and can be performed in tissue and cell samples from most species. Standard staining procedures the internet pathology laboratory for medical education, florida state university college of medicine standardized staining procedures for histology laboratory. Purification of zebrafish erythrocytes as a means of identifying a. Zebrafish module mbl embryology course 2010 june 29th july 3rd manual modified from original version written by marnie halpern and josh gamse, with many contributions from other instructors and tas over the years. It highlights the distinction between neutral lipids triglycerides, cholesteryl esters, steroids which are stained pink and acids fatty acids, chromolipids, phospholipids which are stained blue. May 12, 20 here is the protocol for alcian blue periodic acid schiff ab pas. Lue1,2 the life scienceshoward hughes medical institute outreach program at harvard university supports. It should, however, be noted that ars concentrations used here were much lower 0. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than coomassie staining, and is often as sensitive as silver staining. The protective layer of zebrafish embryo changes continuously.
Clearing and staining for larval fish cartilage and bone. After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml coomassie blue r250 staining solution. The proposed ars vital staining protocol can be combined with gfp. Wholemount staining of biotindextran injected embryos. Useful tip for better zebrafish wholemount ihc staining. Jul 14, 2012 stain gel in the above solution, with 0. Coomassie brillant blue r250 cbr250 and silver staining are the most widely used methods for the routine visualization of proteins separated by sdspage. Jan 10, 2012 zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2.
Not all coomassie stains are compatible with mass spectrometry. Standard protocols for monitoring and sampling zebra mussels. Microwave for 45 sec until the solution just starts to boil. Staining bones with alizarin red s after clearing away the soft tissue. Imaging blood vessels in the zebrafish sciencedirect.
Zebrafish embryology and cartilage staining protocols for. The transparent embryos that develop ex utero make them ideal for assessing cardiac. Protocol for alcian blue staining for frozen sections. Staining protein gels with coomassie blue national diagnostics. Fish collection protocols overview fish as environmental indicators fish community assessments are an important component of many water quality management programs. Calcein, another standard reagent for in vivo skeletal staining of zebrafish, was used as a control staining, following an established calcein staining protocol. Recommended sds page stain protocols kits like gelcode blue from pierce and biosafe coomassie from biorad are not compatible for ingel digestion and. Can you provide a protocol to stain the zebrafish with oil red o to quantify lipids. Photoconversion of fluorescently labeled profiles for em. In alcian blue staining protocol, sf red is used as counter stain which.
Can you provide a protocol to stain the zebrafish with oil. Recent works have used ars vital staining in zebrafish and medaka, yet not based. Skeletal development in zebrafish using alcian bluealizarin red double staining. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. Intestine of ballan wrasse labrus bergylta a species of marine fish was stained with alcian blue pas at ph2. Staining and observation of cartilaginous structures in zebrafish complete larvae introduction. Add 50 ml of 100% ethanol for a final concentration of 10% vv c. We use cookies to provide you with a great user experience. Coomassie brilliant blue g250 differs from coomassie brilliant blue r. Phthalocyanines are synthetic analogues of porphyrins that contain nitrogenaromatic rings surrounding a central atom. Staining membranebound proteins with coomassie blue r250. Skeletal development in zebrafish using alcian bluealizarin red. Which is the best protocol for alcian blue staining and semiquantification in bidimensional cultures.
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. Nile blue is used for histological staining of biological preparations. Monitoring zebra mussels does not require use of all of the techniques describedere. Nov 21, 20 the time required to visualize proteins using coomassie blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a coomassie blue dye solution at boiling temperatures. If any one has a working protocol of oil red o staining pertaining to zebrafish, please share with us. Histological methods protocols zfin community wiki. Alcian blue staining protocol zebrafish best picture of. Coomassie blue staining protocol staining with coomassie blue r250 stain the gel with 0. A twocolor acidfree cartilage and bone stain for zebrafish larvae. Any water stage is a good stopping point if you had to do something part way through the procedure. Prepare metaphase spreads on slides as outlined in a.
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. Protocol for alcian blue staining for micromass cell culture. We accept samples for protein identification from coomassie and silverstained gels. After incubation for 48 h, the medaka, zebrafish, and xenopus became clearer fig. Incubate at room temp with gentle shaking for 1015 min. Check clarity of solution under a microscope to make sure there are no precipitates. Alcian blue 8gx has been used for the staining and precipitation of glycosaminoglycans. Dissolve 25g of aluminum sulfate in 200 ml of millipore water to create a 5% wv concentration b. Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2.
For coomassie brilliant blue staining, intact zebrafish egg of embryonic age 6. After electrophoresis of protein gel, transfer gel to round staining tray. 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. Cells were stained with trypan blue, and living cells were counted using a haematocytometer. Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. A rapid and nondestructive protocol for wholemount bone staining. Carolinablu stain was developed as a significant advance over the methylene blue staining protocol. For example, dechorionated zebrafish embryos at blastulationgastrulation stages. Use caution while performing the following steps using a microwave oven. At the bottom of the post is the link for printing. Cbr250 is an organic dye that complexes with basic amino acids, such as arginine, lysine, and histidine, as well as tyrosine. These assessments are useful for making decisions in regard. Laboratory 6protein isolation from fish and western blotting. This technique is relatively insensitive, with a detection limit of 1.
Zebrafish bone and general physiology are differently affected by. Staining and observation of cartilaginous structures in. Coomassie colloidal staining for overnight stains 1. A 9 dpf zebrafish control larvae head skeleton presenting calcified calcified. Alcian blue stains acid mucosubstances and acetic mucins. Staining of proteins with sypro orange and sypro ruby are also demonstrated here. In the protocol described here, colabeling is limited by the requirement for the specific rabbitderived antibody used in our assay.
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