Adapted from manipulating the mouse embryo, 3rd edition eds. Aug 01, 1984 alcian blue is employed for visualization of the digest fragments. Embryos were dissected from the uterus in 1x phosphate buffer solution pbs and fixed individually in bouins solution for 2 hours at room temperature. Common routine stain not an immunohistochemical stain to detect mucins wikipedia. A combined alcian bluepas stain abpas is frequently used in practice. The alcian blue staining kit contains 1% alcian blue solution, 0. 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. We also include acidfree modifications for staining adult zebrafish. Finally, we propose a novel type of experimental approach, the reduced. To answer the question above, the sound is like alshian not alkian as far as i know it also is consistent with the. Zebrafish embryology and cartilage staining protocols for high. Andras nagy, marina gertsenstein, kristina vintersten and richard behringer. Zebrafish embryology and cartilage staining protocols for. Wash in saturated sodium borate solution for 912 hours.
Alcian blue 8gx can be used to determine proteins based on their resonance light scattering by strongly enhancing the signals. In most protocols, sections are stained with the standard alcian blue ph 2. Alcian bluealizarin red staining was performed as previously described fisher and halpern, 1999. Alcian blue staining protocol zebrafish best picture of. Immunohistochemical staining with alcian blue is a convenient method to determine the extent of sulfation of mucins in tissues. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed bmp2 in zebrafish. 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.
Alcian blue staining of gags in tissue may be modified through the addition of an electrolyte such as magnesium chloride. In the following example, serial sections of the colon from wt and glcnac6st2deficient ko. C close up of head in ventral view of alcian blue cartilage staining at 3dpf. Analysis of dyesubstrate affinities has been carried out using the critical electrolyte concentration cec approach. 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. Pigment cell organization in the hypodermis of zebrafish. 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. 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.
Adapted from manipulating the mouse embryo, 3rd edition, by andras nagy, marina gertsenstein, kristina vintersten, and richard behringer. Cartilage was stained with alcian blue, using a modified protocol of kelly and. 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. Leave the specimen in the alcian blue solution overnight to ensure a good takeup of stain by cartilage. This technique is also useful in diagnosing diseases of the gi tract. This article describes a method for simultaneously visualizing cartilage and mineralized tissues notably bone in the developing mouse. While counterstaining the alcian blue stained cells with fast red, the alcian blue is getting washed off. Alcian blue stain definition of alcian blue stain by. May 16, 2011 the alcian blue stain is this authors preferred stain for the demonstration of acidic mucins. 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.
Clearing and staining for larval fish cartilage and bone. 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. Current methods to visualize mineralized tissue in zebrafish rely on. 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.
The protocol i followed was staining the fixed cells with alcian blue 1% prepared in 3%. Component freshwater algae freshwater fish microtox water flea acetic acid pimephales promelas. Briefly, fish are fixed overnight in 4% paraformaldehydepbs, then stained overnight in 0. 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 work is a continuation of a communication on the stainability of broad bean vicia faba l. A modification of the alcian blue method for staining hyaluronic acid substances in tissue sections. Screening in larval zebrafish reveals tissuespecific distribution of. Alcian blue staining was performed for 30 min in this study. The centra was soaked in alcian blue solution 16 ml 100% ethanol, 2 mg alcian blue and 4 ml glacial acetic acid in 0. A rapid and nondestructive protocol for wholemount bone staining. However, staining cartilage with alcian blue would affect subsequent.
Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation. Before staining a slide, the tissue has to be prepared and mounted onto a glass slide. A guide for the laboratory use of zebrafish danio rerio, eugene, university of oregon press. Ct techniques, that are ultimately limited in resolution. Block and slide identifiers should be clearly written and match exactly with the specimen. Staining was greatest within the pericellular matrix i. Alcian blue and alizarin red staining to evaluate cartilage and bone were performed as previously described 35. Alcian blue staining of the mouse fetal cartilaginous skeleton. Which is the best protocol for alcian blue staining and semi. A alcian blue staining of head cartilage representing the landmarks used for. Remove the fat from the mice but be careful that damage is not done to the specimen. Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. 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. In the fish, preparations of alcian blue can be used to observe individual cartilage from two days postfertilization dpf onwards.
The paraffin technique is the most common way to prepare a histological slide, and follows the following steps. Glucocorticoids alter craniofacial development and increase. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Dat 9dpf the cartilage staining is visible and the jaw becomes prominent arrow. Protocol for alcian blue staining for micromass cell culture. Radiographic analysis of zebrafish skeletal defects. It is used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other. Richardallan scientific alcian bluepas special stain kit. How to enzyme clear and double stain fish specimens. Transfer to two changes of 95% ethyl alcohol, 12 hours per change. At the bottom of the post is the link for printing. Alcian blue staining protocol by roy ellis ihc world.
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 combination of the alcian blue and the pas techniques can be used as a means of distinguishing neutral mucins from acid mucins. Upper and lower panels are dorsal and lateral views, respectively. 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. Sample loads of 510 micrograms per band appear optimum. A modification of the alcian blue method for staining. The ph of alcian blue staining solutions should range from 0. Pas, alcian blue and toluidine blue staining technique. A simple wholemount staining protocol for bone and or cartilage. For this purpose, the alcian blue staining should be performed at ph 1. The dye was originally used for the dyeing of cotton before being discovered as a by steedman in 1950.
After incubation for 48 h, the medaka, zebrafish, and xenopus became. May 12, 20 here is the protocol for alcian blue periodic acid schiff ab pas. Here we describe a novel staining method, designated succinylationalcian blue staining, for visualizing mucins on a pvdf membrane. Can be used alone to stain acid mucins and other carboxylated and sulfated acid mucosubstances blue on tissue sections. Zebrafish bone and general physiology are differently affected by. Cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004. Periodic acid schiff pas reaction is used to stain glycogen, basement membranes and neutral mucosubstances. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde pfa at 4c overnight. Alcian blue stain an overview sciencedirect topics. Embryos to be used for alcian blue staining were grown in 0.
Teleost fish such as zebrafish danio rerio are increasingly used for. On the stainability of plant cell walls with alcian blue. Briefly, the animals were immersion fixed in 10% formalin, followed by. 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. 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. Staining for carbohydratesglycoproteins mucins using alcian bluepas staining for glycogen using pas, pasamylase staining for metachromasia using toluidine blue. Recently acquired data on the complete structure of alcian blue permit interpretations of the affinities in. Product description type unit quantity academic price book. Alcian bluealizarin red staining of cartilage and bone in mouse. Alcian blue alizarin red staining was performed as previously described fisher and halpern, 1999. Pdf alcian bluealizarin red staining of cartilage and bone. 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. Alcian blue staining cartilage was stained with alcian blue using a modified previously published method loucks and carvan, 2004.
A proprietary watersoluble phthalocyanin dye ci 74240 used in histology, which can be modified at different phs to identify specific mucopolysaccharide families. Alcian blue 8gx is a cationic dye used to quantitatively determine glycosaminoglycans by forming insoluble complexes in solution. 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%. Check clarity of solution under a microscope to make sure there are no precipitates. 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. 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. Alcian blue stain for acidic mucins method and tips. Alcian blue staining kit is intended for in vitro diagnostic ivd use as a qualitative histologic stain to demonstrate weakly acidic mucopolysaccharides. Micromass cultures of c3h10t12 cells at varied oxygen tensions stained with alcian blue. Adjusting the ph of alcian blue alters the type of tissue it can bond with. 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 acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone. At this stage the cartilage staining is diffuse and nonspeci.
The smallest oligosaccharide which may be stained by this method is the hexasaccharide. The alcian blue itself is a cationic copper phthalocyanine dye which stains mucopolysaccharides and glycosaminoglycans a bluish colour. Empirical formula hill notation c 56 h 68 cun 16 s 4 4cl. Staining and observation of cartilaginous structures in. Revisiting in vivo staining with alizarin red s a valuable. Alcian blue pas stain protocol histopathology service. Alcian blue alizarin red skeletal staining october 2003 eddy m. The vertebrate skeleton forms by endochondral and intramembranous bone formation. In this study, alcian blue staining technique was used to enhance the visibility of the age bands on vertebrae.
After gradual rehydration, soft tissues are removed by trypsin digestion. Empirical formula hill notation c 56 h 68 cl 4 cun 16 s 4. Age determination with alcian blue staining technique for. 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. Combind alcian blue pas staining technique youtube. Franzodendaal from zebrafish book 5th edition this protocol was originally modified from klymkowski and hanken 1991 for amphibians. Visualizing normal and defective bone development in.
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. Traditionally, cartilage is stained by alcian blue using acidic. Glucocorticoids alter craniofacial development and. Polyacrylamidegel electrophoresis and alcian blue staining.
Mutations affecting craniofacial development in zebrafish. Alcian blue staining for embryos or young larvae 1. Spinal deformity in aged zebrafish is accompanied by. 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. Larvae 5 dpf were euthanized with tricaine then fixed in 5% trichloroacetic acid for 10 min at room temperature followed by rinsing in 0. Packaging 250, 500 ml in glass bottle biochemphysiol actions. 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. Alcian blue will stain acidic mucins blue and pas will stain neutral mucins rose red.1179 501 1140 150 1264 1043 187 492 516 1301 799 960 839 583 812 1521 36 752 281 16 792 1221 575 1052 977 742 664 657 157 278 236 1051 1025 46 141 15