Pas, alcian blue and toluidine blue staining technique. Alcian blue 8gx can be used to determine proteins based on their resonance light scattering by strongly enhancing the signals. 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. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. This kit originally used safranin o solution but this has now been replaced with nuclear fast red solution. Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining.
A proprietary watersoluble phthalocyanin dye ci 74240 used in histology, which can be modified at different phs to identify specific mucopolysaccharide families. Alcian blue staining of gags in tissue may be modified through the addition of an electrolyte such as magnesium chloride. Pdf alcian bluealizarin red staining of cartilage and bone. After gradual rehydration, soft tissues are removed by trypsin digestion. 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. Product description type unit quantity academic price book. Mutations affecting craniofacial development in zebrafish. How to enzyme clear and double stain fish specimens. Leave the specimen in the alcian blue solution overnight to ensure a good takeup of stain by cartilage. C close up of head in ventral view of alcian blue cartilage staining at 3dpf. Screening in larval zebrafish reveals tissuespecific distribution of. Alcian blue alizarin red staining was performed as previously described fisher and halpern, 1999. Radiographic analysis of zebrafish skeletal defects. Adapted from manipulating the mouse embryo, 3rd edition, by andras nagy, marina gertsenstein, kristina vintersten, and richard behringer.
Staining for carbohydratesglycoproteins mucins using alcian bluepas staining for glycogen using pas, pasamylase staining for metachromasia using toluidine blue. Protocol for alcian blue staining for micromass cell culture. At this stage the cartilage staining is diffuse and nonspeci. 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.
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. May 12, 20 here is the protocol for alcian blue periodic acid schiff ab pas. Immunohistochemical staining with alcian blue is a convenient method to determine the extent of sulfation of mucins in tissues. For this purpose, the alcian blue staining should be performed at ph 1. 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. In the fish, preparations of alcian blue can be used to observe individual cartilage from two days postfertilization dpf onwards. Traditionally, cartilage is stained by alcian blue using acidic. Here we describe a novel staining method, designated succinylationalcian blue staining, for visualizing mucins on a pvdf membrane.
A modification of the alcian blue method for staining hyaluronic acid substances in tissue sections. In the following example, serial sections of the colon from wt and glcnac6st2deficient ko. Block and slide identifiers should be clearly written and match exactly with the specimen. Adjusting the ph of alcian blue alters the type of tissue it can bond with. Transfer to two changes of 95% ethyl alcohol, 12 hours per change.
Glucocorticoids alter craniofacial development and increase. Aug 01, 1984 alcian blue is employed for visualization of the digest fragments. Adapted from manipulating the mouse embryo, 3rd edition eds. Alcian bluealizarin red staining of cartilage and bone in. Alcian blue stain an overview sciencedirect topics. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. Alcian blue stain for acidic mucins method and tips. A modification of the alcian blue method for staining. 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. Common routine stain not an immunohistochemical stain to detect mucins wikipedia. Can be used alone to stain acid mucins and other carboxylated and sulfated acid mucosubstances blue on tissue sections. After incubation for 48 h, the medaka, zebrafish, and xenopus became. A simple wholemount staining protocol for bone and or cartilage.
The combination of the alcian blue and the pas techniques can be used as a means of distinguishing neutral mucins from acid mucins. In most protocols, sections are stained with the standard alcian blue ph 2. Finally, we propose a novel type of experimental approach, the reduced. Red and alcian bluestained zebrafish larva at 14 dpf. The tissue sample is resectioned and fixed upon a slide. Alcian blue staining protocol by roy ellis ihc world. The ph of alcian blue staining solutions should range from 0. Teleost fish such as zebrafish danio rerio are increasingly used for. 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. This technique is also useful in diagnosing diseases of the gi tract. Analysis of dyesubstrate affinities has been carried out using the critical electrolyte concentration cec approach. Alcian blue stain definition of alcian blue stain by.
Alcian blue 8gx is a cationic dye used to quantitatively determine glycosaminoglycans by forming insoluble complexes in solution. Cartilage was stained with alcian blue, using a modified protocol of kelly and. The alcian blue staining kit contains 1% alcian blue solution, 0. 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. Revisiting in vivo staining with alizarin red s a valuable. 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. 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. 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. Alcian blue staining protocol zebrafish best picture of. Staining and observation of cartilaginous structures in.
The alcian blue itself is a cationic copper phthalocyanine dye which stains mucopolysaccharides and glycosaminoglycans a bluish colour. Component freshwater algae freshwater fish microtox water flea acetic acid pimephales promelas. Current methods to visualize mineralized tissue in zebrafish rely on. Embryos to be used for alcian blue staining were grown in 0. 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 pas stain protocol histopathology service. 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. Cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004. The centra was soaked in alcian blue solution 16 ml 100% ethanol, 2 mg alcian blue and 4 ml glacial acetic acid in 0. Alcian bluealizarin red staining was performed as previously described fisher and halpern, 1999.
Alcian blue staining for embryos or young larvae 1. Before staining a slide, the tissue has to be prepared and mounted onto a glass slide. This work is a continuation of a communication on the stainability of broad bean vicia faba l. At the bottom of the post is the link for printing. Alcian blue staining cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004.
Alcian blue alizarin red skeletal staining october 2003 eddy m. On the stainability of plant cell walls with alcian blue. Pigment cell organization in the hypodermis of zebrafish. Zebrafish embryology and cartilage staining protocols for. Remove the fat from the mice but be careful that damage is not done to the specimen. In this study, alcian blue staining technique was used to enhance the visibility of the age bands on vertebrae. Alcian blue and alizarin red staining to evaluate cartilage and bone were performed as previously described 35. Briefly, the animals were immersion fixed in 10% formalin, followed by. Embryos were dissected from the uterus in 1x phosphate buffer solution pbs and fixed individually in bouins solution for 2 hours at room temperature. The protocol i followed was staining the fixed cells with alcian blue 1% prepared in 3%. Clearing and staining for larval fish cartilage and bone. Richardallan scientific alcian bluepas special stain kit.
Periodic acid schiff pas reaction is used to stain glycogen, basement membranes and neutral mucosubstances. Empirical formula hill notation c 56 h 68 cl 4 cun 16 s 4. This article describes a method for simultaneously visualizing cartilage and mineralized tissues notably bone in the developing mouse. 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. The dye was originally used for the dyeing of cotton before being discovered as a by steedman in 1950.
While counterstaining the alcian blue stained cells with fast red, the alcian blue is getting washed off. 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. Sample loads of 510 micrograms per band appear optimum. Spinal deformity in aged zebrafish is accompanied by. 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. Age determination with alcian blue staining technique for. Dissect mice by removing the skin and organs completely. Alcian bluealizarin red staining of cartilage and bone in mouse.
However, staining cartilage with alcian blue would affect subsequent. 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. A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press. Zebrafish embryology and cartilage staining protocols for high. Combind alcian blue pas staining technique youtube. Alcian blue staining kit is intended for in vitro diagnostic ivd use as a qualitative histologic stain to demonstrate weakly acidic mucopolysaccharides. A twocolor acidfree cartilage and bone stain for zebrafish larvae. Alcian blue staining of the mouse fetal cartilaginous skeleton.
Recently acquired data on the complete structure of alcian blue permit interpretations of the affinities in. It is used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other. The paraffin technique is the most common way to prepare a histological slide, and follows the following steps. 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. A alcian blue staining of head cartilage representing the landmarks used for. Empirical formula hill notation c 56 h 68 cun 16 s 4 4cl. Alcian blue will stain acidic mucins blue and pas will stain neutral mucins rose red. 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. May 16, 2011 the alcian blue stain is this authors preferred stain for the demonstration of acidic mucins. Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed bmp2 in zebrafish. 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.
Jan 19, 2016 the correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Visualizing normal and defective bone development in. Which is the best protocol for alcian blue staining and semi. Ct techniques, that are ultimately limited in resolution. 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. 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. Packaging 250, 500 ml in glass bottle biochemphysiol actions. Andras nagy, marina gertsenstein, kristina vintersten and richard behringer. Briefly, fish are fixed overnight in 4% paraformaldehydepbs, then stained overnight in 0.
Alcian blue is a dye used to detect cell chondrogenesis and it stains the sulfated proteoglycan in cartilage tissue. Im having trouble with facial cartilage staining in zebrafish 120hpf. A rapid and nondestructive protocol for wholemount bone staining. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow. Alcian blue staining was performed for 30 min in this study. Micromass cultures of c3h10t12 cells at varied oxygen tensions stained with alcian blue. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone.
A simple wholemount staining protocol for bone and or. Larvae 5 dpf were euthanized with tricaine then fixed in 5% trichloroacetic acid for 10 min at room temperature followed by rinsing in 0. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. A combined alcian bluepas stain abpas is frequently used in practice. 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. 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%. 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. 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. We also include acidfree modifications for staining adult zebrafish. Staining was greatest within the pericellular matrix i. 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.
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