A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press. 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. Upper and lower panels are dorsal and lateral views, respectively. Franzodendaal from zebrafish book 5th edition this protocol was originally modified from klymkowski and hanken 1991 for amphibians.
The paraffin technique is the most common way to prepare a histological slide, and follows the following steps. Combind alcian blue pas staining technique youtube. Embryos were euthanatized using an overdose of ms222, fixed overnight at 4c in 4% paraformaldehyde, and then transferred to 70% ethanol to allow dehydration of the tissue for at least 24 h. Mutations affecting craniofacial development in zebrafish. Jan 19, 2016 the correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. May 12, 20 here is the protocol for alcian blue periodic acid schiff ab pas. Dissect mice by removing the skin and organs completely. The ph of the alcian blue staining solution is a critical factor in the staining procedure and has a direct effect on the category of mucins that can be demonstrated with the procedure.
Screening in larval zebrafish reveals tissuespecific distribution of. Place directly into a mixture of 10 mg alcian blue 8gn supplied by matheson, coleman and bell, 80 ml 95% ethyl alcohol and 20 ml glacial acetic acid 1012 hours. 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. The vertebrate skeleton forms by endochondral and intramembranous bone formation. A twocolor acidfree cartilage and bone stain for zebrafish larvae. In the following example, serial sections of the colon from wt and glcnac6st2deficient ko. This kit originally used safranin o solution but this has now been replaced with nuclear fast red solution. Transfer to two changes of 95% ethyl alcohol, 12 hours per change. A alcian blue staining of head cartilage representing the landmarks used for. Embryos were dissected from the uterus in 1x phosphate buffer solution pbs and fixed individually in bouins solution for 2 hours at room temperature. On the stainability of plant cell walls with alcian blue.
Sample loads of 510 micrograms per band appear optimum. Ct techniques, that are ultimately limited in resolution. How to enzyme clear and double stain fish specimens. With any luck, you will need to collect only one ribbon containing gills one bisected half and spinal cord the other bisected half, along with a nice sampling of internal organs. Alcian blue will stain acidic mucins blue and pas will stain neutral mucins rose red.
Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. Zebrafish embryology and cartilage staining protocols for high. 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. Age determination with alcian blue staining technique for. Combind alcian blue pas staining technique,pas,staining technique,alcian blue pas staining technique,pas staining,histology staining,histology crash course,histology staining techniques,histology. Aug 01, 1984 alcian blue is employed for visualization of the digest fragments. Im having trouble with facial cartilage staining in zebrafish 120hpf. For this purpose, the alcian blue staining should be performed at ph 1. To answer the question above, the sound is like alshian not alkian as far as i know it also is consistent with the. Alcian blue 8gx can be used to determine proteins based on their resonance light scattering by strongly enhancing the signals. Alcian blue 8gx is a cationic dye used to quantitatively determine glycosaminoglycans by forming insoluble complexes in solution. Briefly, the animals were immersion fixed in 10% formalin, followed by.
This article describes a method for simultaneously visualizing cartilage and mineralized tissues notably bone in the developing mouse. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow. Staining was greatest within the pericellular matrix i. Alcian blue staining kit is intended for in vitro diagnostic ivd use as a qualitative histologic stain to demonstrate weakly acidic mucopolysaccharides.
Briefly, fish are fixed overnight in 4% paraformaldehydepbs, then stained overnight in 0. Alcian blue staining was performed for 30 min in this study. Radiographic analysis of zebrafish skeletal defects. Glucocorticoids alter craniofacial development and.
Pigment cell organization in the hypodermis of zebrafish. Alcian blue ln is any member of a family of polyvalent basic dyes, of which the alcian blue 8g also called ingrain blue 1, and c. After incubation for 48 h, the medaka, zebrafish, and xenopus became. The rationale of the technique is that by first staining all the acidic mucins with alcian blue, those remaining acidic mucins which are also pas positive will be chemically blocked and will not react further during the technique.
Zebrafish bone and general physiology are differently affected by. Spinal deformity in aged zebrafish is accompanied by. A simple wholemount staining protocol for bone and or cartilage. Larvae 5 dpf were euthanized with tricaine then fixed in 5% trichloroacetic acid for 10 min at room temperature followed by rinsing in 0. Alcian blue and alizarin red staining to evaluate cartilage and bone were performed as previously described 35. In most protocols, sections are stained with the standard alcian blue ph 2. Recently acquired data on the complete structure of alcian blue permit interpretations of the affinities in. Adjusting the ph of alcian blue alters the type of tissue it can bond with. Alcian blue stain an overview sciencedirect topics. Protocol for alcian blue staining for micromass cell culture. The combination of the alcian blue and the pas techniques can be used as a means of distinguishing neutral mucins from acid mucins. Alcian bluealizarin red staining of cartilage and bone in mouse. A rapid and nondestructive protocol for wholemount bone staining.
This work is a continuation of a communication on the stainability of broad bean vicia faba l. Alcian blue may be used in combination with the pas staining procedure, so that both acid and neutral mucins can be demonstrated in the same tissue sample. Here we describe a novel staining method, designated succinylationalcian blue staining, for visualizing mucins on a pvdf membrane. In this study, alcian blue staining technique was used to enhance the visibility of the age bands on vertebrae. Visualizing normal and defective bone development in. Cartilage was stained with alcian blue, using a modified protocol of kelly and. However, staining cartilage with alcian blue would affect subsequent. The tissue sample is resectioned and fixed upon a slide. Alcian bluealizarin red staining of cartilage and bone in. Adapted from manipulating the mouse embryo, 3rd edition eds. The alcian blue itself is a cationic copper phthalocyanine dye which stains mucopolysaccharides and glycosaminoglycans a bluish colour. Histological staining of midsagittal sections through fish at each age with alcian blue identified the gagrich, cartilaginous inner facet of the vertebral body figure 2a,b. This protocol was originally modified from klymkowski and hanken 1991 for amphibians.
To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed bmp2 in zebrafish. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei except nucleolus lightly, the nucleolus and cytoplasm are practically colourless. A modification of the alcian blue method for staining. Revisiting in vivo staining with alizarin red s a valuable.
Current methods to visualize mineralized tissue in zebrafish rely on. 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. Staining and observation of cartilaginous structures in. Alcian blue pas stain protocol histopathology service. It is used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other. To answer the question above, the sound is like alshian not alkian as far as i know it also is consistent with the history of the name supposedly coming from phthallocyanine. Alcian blue is a dye used to detect cell chondrogenesis and it stains the sulfated proteoglycan in cartilage tissue. Red and alcian bluestained zebrafish larva at 14 dpf. Alcian blue alizarin red staining was performed as previously described fisher and halpern, 1999. Block and slide identifiers should be clearly written and match exactly with the specimen. Empirical formula hill notation c 56 h 68 cl 4 cun 16 s 4. A proprietary watersoluble phthalocyanin dye ci 74240 used in histology, which can be modified at different phs to identify specific mucopolysaccharide families.
The protocol i followed was staining the fixed cells with alcian blue 1% prepared in 3%. Leave the specimen in the alcian blue solution overnight to ensure a good takeup of stain by cartilage. Alcian blue stain definition of alcian blue stain by. Empirical formula hill notation c 56 h 68 cun 16 s 4 4cl. While counterstaining the alcian blue stained cells with fast red, the alcian blue is getting washed off. Component freshwater algae freshwater fish microtox water flea acetic acid pimephales promelas.
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. The dye was originally used for the dyeing of cotton before being discovered as a by steedman in 1950. Teleost fish such as zebrafish danio rerio are increasingly used for. In the fish, preparations of alcian blue can be used to observe individual cartilage from two days postfertilization dpf onwards.
The alcian blue staining kit contains 1% alcian blue solution, 0. Can be used alone to stain acid mucins and other carboxylated and sulfated acid mucosubstances blue on tissue sections. Alcian blue staining of the mouse fetal cartilaginous skeleton. Alcian blue staining protocol by roy ellis ihc world. The cartilaginous skeleton is stained dark blue by alcian blue, whereas the other tissues of the embryo are made transparent by a clearing solution, benzyl alcoholbenzyl benzoate babb. A modification of the alcian blue method for staining hyaluronic acid substances in tissue sections. Packaging 250, 500 ml in glass bottle biochemphysiol actions. Polyacrylamidegel electrophoresis and alcian blue staining. The rationale of the technique is that by first staining all the acidic mucins with alcian blue, those remaining acidic mucins which are also pas positive will be. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon older literature referred to zebrafish as brachydanio rerio.
May 16, 2011 the alcian blue stain is this authors preferred stain for the demonstration of acidic mucins. Analysis of dyesubstrate affinities has been carried out using the critical electrolyte concentration cec approach. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde pfa at 4c overnight. Product description type unit quantity academic price book. 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. Before staining a slide, the tissue has to be prepared and mounted onto a glass slide. A simple wholemount staining protocol for bone and or. Alcian blue staining cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004. At this stage the cartilage staining is diffuse and nonspeci. Immunohistochemical staining with alcian blue is a convenient method to determine the extent of sulfation of mucins in tissues. Alcian blue staining for embryos or young larvae 1. Wash in saturated sodium borate solution for 912 hours. Which is the best protocol for alcian blue staining and semi. Pdf alcian bluealizarin red staining of cartilage and bone.
Clearing and staining for larval fish cartilage and bone. Common routine stain not an immunohistochemical stain to detect mucins wikipedia. Richardallan scientific alcian bluepas special stain kit. Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. It makes use of alcian blue and alizarin red stains, and it is best used for later fetal, newborn, and early postnatal stages of development. Glucocorticoids alter craniofacial development and increase.
Finally, we propose a novel type of experimental approach, the reduced. Check clarity of solution under a microscope to make sure there are no precipitates. Cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone. Traditionally, cartilage is stained by alcian blue using acidic. Because the fish has been cut to one side of the spinal cord, you will be cutting toward the midline on one half of the fish, and away from the midline on the other. 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 structures. Embryos to be used for alcian blue staining were grown in 0. Alcian blue staining of gags in tissue may be modified through the addition of an electrolyte such as magnesium chloride.
C close up of head in ventral view of alcian blue cartilage staining at 3dpf. Alcian blue staining protocol zebrafish best picture of. Andras nagy, marina gertsenstein, kristina vintersten and richard behringer. Micromass cultures of c3h10t12 cells at varied oxygen tensions stained with alcian blue. The ph of alcian blue staining solutions should range from 0. Contents from the alcian blue is now moved here to this staining entry, with this page redirected here and a link to the did you mean the alcian blue band. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. At the bottom of the post is the link for printing. Remove the fat from the mice but be careful that damage is not done to the specimen. Alcian bluealizarin red staining was performed as previously described fisher and halpern, 1999. Alcian blue alizarin red skeletal staining october 2003 eddy m.
We also include acidfree modifications for staining adult zebrafish. The centra was soaked in alcian blue solution 16 ml 100% ethanol, 2 mg alcian blue and 4 ml glacial acetic acid in 0. Pas, alcian blue and toluidine blue staining technique. After gradual rehydration, soft tissues are removed by trypsin digestion. Periodic acid schiff pas reaction is used to stain glycogen, basement membranes and neutral mucosubstances. Alcian blue stain for acidic mucins method and tips. Staining for carbohydratesglycoproteins mucins using alcian bluepas staining for glycogen using pas, pasamylase staining for metachromasia using toluidine blue. Zebrafish embryology and cartilage staining protocols for. This technique is also useful in diagnosing diseases of the gi tract. A combined alcian bluepas stain abpas is frequently used in practice. Large intestine deparaffin slides in xylene 1 2 minutes reuse deparaffin slides in xylene 2 2 minutes reuse clear slides in 100% alcohol 2 minutes clear slides in 100%.
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