At the bottom of the post is the link for printing. In modern anatomy, there are no threadlike structures floating inside blood vessels without adhering to the vessel wall. Alcian blue is a dye used to detect cell chondrogenesis and it stains the sulfated proteoglycan in cartilage tissue. A rapid and nondestructive protocol for wholemount bone. Zebrafish embryology and cartilage staining protocols for. The alcian blue staining kit contains 1% alcian blue solution, 0. The protocol i followed was staining the fixed cells with alcian blue 1% prepared in 3%. Alcian blue stain for acidic mucins method and tips. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow.
Zebrafish embryology and cartilage staining protocols for high. While counterstaining the alcian blue stained cells with fast red, the alcian blue is getting washed off. Current methods to visualize mineralized tissue in. Cartilage was stained with alcian blue, using a modified protocol of kelly and. However recently we found novel threadlike tissues that are called.
Common routine stain not an immunohistochemical stain to detect mucins wikipedia. Hito alcian blue pas optimstain kit hitobiotec corp. Upper and lower panels are dorsal and lateral views, respectively. Alcian blue staining at acidic ph is widely used as an indicator of the presence of. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration.
Richardallan scientific alcian bluepas special stain kit. Briefly, the animals were immersion fixed in 10% formalin, followed by. Committee and followed the national institutes of health guidelines for the care and use. The chemical protocol was inspired by fleming and collaborators experiments 63. The dye was originally used for the dyeing of cotton before being discovered as a by. A alcian blue staining of head cartilage representing the landmarks. Calcein, another standard reagent for in vivo skeletal staining of zebrafish, was used as a control staining, following an established calcein staining protocol. Go to the method 3 alcian blue cartilage stain protocol, starting at step 2. Alcian blue and alizarin red staining to evaluate cartilage and bone were performed as previously described 35. Alcian blue can be used to quantitate acidic glycans both in microspectrophotometric quantitation in solution or for staining glycoproteins in polyacrylamide gels or on western blots.
Alizarin red s binds to calcium and so stains selectively for the presence of ossified bone only. Screening in larval zebrafish reveals tissuespecific distribution of. Glucocorticoids alter craniofacial development and. Here is the protocol for alcian blue periodic acid schiff ab pas. Which is the best protocol for alcian blue staining and. A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press.
The method presented in this report offers many advantages over the schiffperiodic acid method and its modification for the staining of. Red and alcian bluestained zebrafish larva at 14 dpf. Protocol for alcian blue staining for frozen sections. Compared with alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal. Alcian blue stain an overview sciencedirect topics. Finally, we propose a novel type of experimental approach, the. Any water stage is a good stopping point if you had to do something part way through. Two new cationic copper phthalocyanins, isomers of each other, have been synthesised, and compared with alcian blue 8gx as stains for a variety of polyanions.
Cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004. However, staining cartilage with alcian blue would affect subsequent. Larvae 5 dpf were euthanized with tricaine then fixed in 5%. 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. Typical examples of glycoprotein patterns are depicted in fig. Alcian blue staining method for visualizing bonghan ducts. Alcian blue pas stain kit from atom scientific stain. Visualizing normal and defective bone development in.
Unlabeled control a and gadofluorinemcylabeled pellets b show stronger staining for intracellular glycosaminoglycan gag. Zebrafish bone and general physiology are differently affected by. Hito alcian blue pas optimstain kit has been tested extensively on the. Clearing and staining for larval fish cartilage and bone. Alcian blue staining for embryos or young larvae 1.
Embryos to be used for alcian blue staining were grown in 0. Fill a 250 ml tissue culture flask with 150 ml of sterile autoclaved fish system water. The most commonly used stains forfetal double staining are alizarin red s andalcian blue. Alcian bluealizarin red staining of cartilage and bone in. Protocol for alcian blue staining for micromass cell culture. Experiments of cell proliferation and differentiation on titanium matrices m17. Can be used alone to stain acid mucins and other carboxylated and sulfated acid mucosubstances blue on tissue sections.
Mutations affecting craniofacial development in zebrafish. The advantage of using alcian blue is it is easy to apply. Teleost fish such as zebrafish danio rerio are increasingly used for physiological. A simple wholemount staining protocol for bone and or cartilage. In the fish, preparations of alcian blue can be used to observe individual cartilage from two days postfertilization dpf onwards. Alcian blue staining protocol zebrafish best picture of.
Alcian blue pas stain protocol histopathology service. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde pfa at 4c overnight. Check clarity of solution under a microscope to make sure there are no precipitates. Traditionally, cartilage is stained by alcian blue using acidic conditions. 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. Protocol for alcian blue staining on whole mount embryos. How to enzyme clear and double stain fish specimens. Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. What is difference between safranino and alcian blue. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Hito alcian blue pas optimstain kit is designed in a readytouse format and offers high quality, rapid and reproducible staining. Alcian bluealizarin red staining of cartilage and bone in mouse. Another common method, still used today, is a doublestaining procedure in which differential staining of cartilage with alcian blue is combined with bone staining with alizarin red s 3.
Large intestine deparaffin slides in xylene 1 2 minutes. Embryos were dissected from the uterus in 1x phosphate buffer solution pbs and fixed individually in. Alcian blue stains acid mucosubstances and acetic mucins. Staining and observation of cartilaginous structures in. C close up of head in ventral view of alcian blue cartilage staining at 3dpf. It should, however, be noted that ars concentrations used here were much lower 0. Various fixatives have been applied and tested for single or doublestaining bone andor cartilage, including ethanol, bouins fixative, a mixture of acetic acid and ethanol, formalin, and paraformaldehyde 3 5. Poly scientific k09816oz alcian blue pas method encompass catalog no.
A twocolor acidfree cartilage and bone stain for zebrafish larvae. A combined alcian bluepas stain abpas is frequently used in. At this stage the cartilage staining is diffuse and nonspeci. Adjusting the ph of alcian blue alters the type of tissue it can bond with. Alcian blue staining of glycoproteins in acrylamide disc. A rapid and nondestructive protocol for wholemount bone staining. Regain access you can regain access to a recent pay per article purchase if your access. Adapted from manipulating the mouse embryo, 3rd edition eds. The alcian blue stain is this authors preferred stain for the demonstration of acidic mucins. After incubation for 48 h, the medaka, zebrafish, and xenopus became. Periodic acid schiff pas reaction is used to stain glycogen, basement. Excessive amounts of nonsulfated acidic mucosubstances are. As the name implies, it entails cutting samples of body.
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