Typical examples of glycoprotein patterns are depicted in fig. A rapid and nondestructive protocol for wholemount bone. Clearing and staining for larval fish cartilage and bone. Hito alcian blue pas optimstain kit has been tested extensively on the. Zebrafish bone and general physiology are differently affected by. Alcian blue staining protocol zebrafish best picture of. Alcian blue is a dye used to detect cell chondrogenesis and it stains the sulfated proteoglycan in cartilage tissue. At the bottom of the post is the link for printing. Check clarity of solution under a microscope to make sure there are no precipitates. C close up of head in ventral view of alcian blue cartilage staining at 3dpf.
Alcian blue and alizarin red staining to evaluate cartilage and bone were performed as previously described 35. Cartilage was stained with alcian blue, using a modified protocol of kelly and. A twocolor acidfree cartilage and bone stain for zebrafish larvae. 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. Alcian blue staining protocol by roy ellis ihc world, llc. A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press. Zebrafish embryology and cartilage staining protocols for high. Poly scientific k09816oz alcian blue pas method encompass catalog no. Im having trouble with facial cartilage staining in zebrafish 120hpf. Glucocorticoids alter craniofacial development and. 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. A combined alcian bluepas stain abpas is frequently used in. Zebrafish embryology and cartilage staining protocols for.
Adapted from manipulating the mouse embryo, 3rd edition eds. The chemical protocol was inspired by fleming and collaborators experiments 63. The alcian blue staining kit contains 1% alcian blue solution, 0. Committee and followed the national institutes of health guidelines for the care and use. Hito alcian blue pas optimstain kit is designed in a readytouse format and offers high quality, rapid and reproducible staining. Alcian blue staining method for visualizing bonghan ducts. The method presented in this report offers many advantages over the schiffperiodic acid method and its modification for the staining of. In the fish, preparations of alcian blue can be used to observe individual cartilage from two days postfertilization dpf onwards. After incubation for 48 h, the medaka, zebrafish, and xenopus became. Protocol for alcian blue staining for frozen sections.
Can be used alone to stain acid mucins and other carboxylated and sulfated acid mucosubstances blue on tissue sections. Alcian blue staining at acidic ph is widely used as an indicator of the presence of. A alcian blue staining of head cartilage representing the landmarks. Alizarin red s binds to calcium and so stains selectively for the presence of ossified bone only. Alcian blue stains acid mucosubstances and acetic mucins. Large intestine deparaffin slides in xylene 1 2 minutes. Fill a 250 ml tissue culture flask with 150 ml of sterile autoclaved fish system water. Alcian blue stain for acidic mucins method and tips. Embryos were dissected from the uterus in 1x phosphate buffer solution pbs and fixed individually in. Common routine stain not an immunohistochemical stain to detect mucins wikipedia. It should, however, be noted that ars concentrations used here were much lower 0. Upper and lower panels are dorsal and lateral views, respectively.
How to enzyme clear and double stain fish specimens. 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. Experiments of cell proliferation and differentiation on titanium matrices m17. Protocol for alcian blue staining for micromass cell culture. The most commonly used stains forfetal double staining are alizarin red s andalcian blue.
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. Protocol for alcian blue staining on whole mount embryos. Embryos to be used for alcian blue staining were grown in 0. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde pfa at 4c overnight. At this stage the cartilage staining is diffuse and nonspeci. Screening in larval zebrafish reveals tissuespecific distribution of.
What is difference between safranino and alcian blue. Finally, we propose a novel type of experimental approach, the. Alcian blue pas stain protocol histopathology service. 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. Larvae 5 dpf were euthanized with tricaine then fixed in 5%. While counterstaining the alcian blue stained cells with fast red, the alcian blue is getting washed off. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. A simple wholemount staining protocol for bone and or cartilage.
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. Here is the protocol for alcian blue periodic acid schiff ab pas. In modern anatomy, there are no threadlike structures floating inside blood vessels without adhering to the vessel wall. 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. Richardallan scientific alcian bluepas special stain kit. Briefly, the animals were immersion fixed in 10% formalin, followed by. Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. Staining and observation of cartilaginous structures in. The advantage of using alcian blue is it is easy to apply. Visualizing normal and defective bone development in. A rapid and nondestructive protocol for wholemount bone staining.
The alcian blue stain is this authors preferred stain for the demonstration of acidic mucins. Mutations affecting craniofacial development in zebrafish. Periodic acid schiff pas reaction is used to stain glycogen, basement. Alcian bluealizarin red staining of cartilage and bone in mouse. Unlabeled control a and gadofluorinemcylabeled pellets b show stronger staining for intracellular glycosaminoglycan gag. However, staining cartilage with alcian blue would affect subsequent. However recently we found novel threadlike tissues that are called. The dye was originally used for the dyeing of cotton before being discovered as a by. Alcian bluealizarin red staining of cartilage and bone in. Which is the best protocol for alcian blue staining and. Alcian blue staining technique is widely used among developmental biologists to observe the embryonic development of cartilage and bone structures in embryos and complete larvae. Traditionally, cartilage is stained by alcian blue using acidic conditions. Any water stage is a good stopping point if you had to do something part way through.
The protocol i followed was staining the fixed cells with alcian blue 1% prepared in 3%. Red and alcian bluestained zebrafish larva at 14 dpf. Teleost fish such as zebrafish danio rerio are increasingly used for physiological. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow. Go to the method 3 alcian blue cartilage stain protocol, starting at step 2. Alcian blue staining of glycoproteins in acrylamide disc. Alcian blue staining for embryos or young larvae 1. Adjusting the ph of alcian blue alters the type of tissue it can bond with. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Alcian blue stain an overview sciencedirect topics. Hito alcian blue pas optimstain kit hitobiotec corp. Excessive amounts of nonsulfated acidic mucosubstances are. Alcian blue pas stain kit from atom scientific stain. Cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004.
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