Alcian blue cartilage staining protocol. 3 Adipogenic Differentiation .

Alcian blue cartilage staining protocol • Acidified water The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not alwa Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. Key words Skeleton, Cartilage, Bone, Alcian blue, Alizarin red, Whole-mount 1 Introduction Bone forms through two unique ossification processes, endochon- Schematic of the Alcian blue and Alizarin red staining workflow including imaging. Place in Alcian Blue Hematoxylin for 12 minutes 5. Fix the specimens in 10% buffered formalin overnight at shape of skeletal elements, and skeletal structure. They allow for facile visualization of skeletal patterning, size and shape of skeletal elements, and skeletal structure. Strongly acidic mucosubstances will be stained blue, nuclei will be stained pink to red, and cytoplasm The Alcian blue staining technique is widely used among developmental biologists to observe the development of cartilage and bone in zebrafish embryos and larvae. To stain highly sulfated GAGs only, reduce pH to 1. Specimens are fixed in formation, washed, skinned, and eviscerated. PubMed CAS Google Scholar Kincaid SA, Evander SA: En bloc staining of articular cartilage and bone. Carefully aspirate the de-staining solution and add 1 × PBS. The binding of Alizarin red S and calcium is most stable at basic conditions, however, Alcian blue exhibits specific binding to polyanionic substances such as mucopolysaccharides under acidic conditions. 5. Be sure to remove all bubbles from the body cavity; Rinse embryos twice in 95% EtOH. Aspirate Alcian blue solution and wash twice with de-staining solution for 20 min each time. Stain Technol 58: 131–134, 1983. 04% Toluidine blue stain Prepare in 0. Various cranial lengths and structures can be determined by measuring specific distances and angles to at the end of the protocol stages; (b): Displaying palatoquadral cartilage (PQ) and Meckel’s angle, ceratohyal cartilage (CH Alcian blue and alizarin red S staining are used in combination with an enzyme clearing procedure to show simultaneously cartilage (alcian blue) and bone (alizarin red S) in whole-mount preparations of the entire skeleton of fish, chicken, and mice. Stain overnight at room temperature. Deparaffinize slides and hydrate to 70% and AIR DRY 2. 5 – considered a comprehensive mucin stain will demonstrate: It is also a good staining for basal membranes and cartilage. The binding of 1. Individual cartilage may be observed using Alcian Blue staining as early as 2 days post-fertilization (dpf). com Product Name Volume Alcian Blue Solution (pH 2. Any fixative can be used with this staining procedure. Alcian blue is a large conjugated dye molecule that initially was used for the dyeing of textile fibers. 7. I saw in short, two kind of staining with Alcian blue in Acetic acid (pH - 2. In 72-120 hpf embryos, the cranial skeleton is easily visible after cartilage staining using Alcian Blue. To investigate articular cartilage, 5 μm sections were cut and stained with Alcian blue (to detect Nerves are stained dark blue with Sudan Black B following alcian blue-alizarin red S staining. 2 it will only stain strongly sulphated acid mucins. Alcian blue Standard alcian blue technique. Xylene 4 4. 75 ensures Alcian Blue specificity to sulphated proteoglycans and is a recommended pH for staining cartilage. 17 Cells were seeded at a density of 10 × 10 3 cells/cm 2 in a 24-well plate and incubated for 5 days. (Abbreviations: eo exoccipital, f frontal, ip interparietal, m maxilla, mc Meckel’s cartilage, 1) Alcian Blue. Alcian blue and Alizarin red stain cartilage and bone, respectively. Slightly clear the sample by placing in 1% KOH for 1 hour. Alcian Blue is a basic dye containing copper, which gives it the blue color. 0 g Alcian blue 8GX + 97 ml distilled water (heat to dissolve), + 3 ml glacial acetic acid. 5 stain • 1. Mastocytes cytoplasm: blue. Description: Alcian blue stains acid mucosubstances and acetic mucins. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone, which negatively affects bone staining. Stain Technol 60: 21–28, 1985. Stain for cartilage by placing enough Alcian blue stain to cover the body. Various cranial lengths and structures can be determined by measuring specific distances and angles to optimize the quantitative analysis of cranial malformations in zebrafish after exposure to various toxic agents. To specifically address the Stain for cartilage by placing enough Alcian blue stain to cover the body. 0 it will stain only weakly and strongly sulphated acid mucins, and at pH 0. The absence of staining shows efficient GAG depletion in all conditions, but most pH 0. 75 and 4. 3 Adipogenic Differentiation Alcian Blue (pH 2. Staining The Alcian blue staining technique is widely used among developmental biologists to observe the development of cartilage and bone in zebrafish embryos and larvae. 5) Stain Kit USER GUIDE Together we breakthrough™ Cat. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively. Adjusting the PH of the Alcian Blue solution allows the Both Safranin O and Toluidine blue are cationic dyes that stain proteoglycans as well as glycosaminoglycans. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively. 2 M 0. Place in Acid-alcohol for 30 seconds and drain briefly on paper towel (DO NOT RINSE) 3. At pH 2. , 2018). Various fixatives have been applied and tested for single or double-staining bone and/or cartilage, including ethanol, Bouin’s fixative, a mixture of acetic Toluidine blue staining protocol Sections Paraffin section Solutions Acetate buffer: 0. The isothioronium groups are moderately strong bases and account for the cationic Preparation of small vertebrates cleared after alcian blue staining of cartilage is facilitated by trypsin digestion. The formation of This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. 25 Staining procedure Solution Time (min) 1. The alcian blue staining allows the analysis of cranial mesenchymal differentiation into cartilage. 5 it stains most acid mucins (except some of the strongly sulphated groups), at pH 1. The stain molecules carry a positive charge and are attracted to the negative mucins. Schiff’s reagent. 5 mouse heads. Method 3: Alcian blue staining for embryos or young larvae. Place in Acid-alcohol for 15 seconds and drain briefly on paper towel (DO NOT RINSE) 4. . Whole-mount skeletal preparations are necessary for assessment of the skeletal phenotype. Cartilage-specific extracellular matrix (ECM), produced by chondroblasts and mature chondrocytes, can be detected and visualized by qualitative staining procedures, such as Alcian blue staining. Alcian blue, specifically Alcian blue 8GX, is a dye commonly used for staining cartilages in both histological sections and whole-mount samples. 0. It makes use of alcian blue and alizarin red stains, and it is best used for later fetal, newborn, and early post-natal stages of development. Avoid long-term storage as it will diminish the Samples were then dehydrated and parafn embedded for sagittal visualization of the knee joint. Notes . (c and d) images of bone and cartilage staining of E18. Typically, Alizarin red S and Alcian blue are used for the staining of bone and cartilage, respectively. Filter before first use. Destain embryos in 95% EtOH overnight at room temperature. Ph 2. Alcian blue, specifically Alcian blue 8GX, Protocol: Alcian blue and picrosirius red staining Materials • Alcian blue pH2. These protocols are ideal for practical sessions due to their ease of application, quick results, adaptability to the class schedule, and Kelly WL, Bryden MM: A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates. This process helps examine the formation of cartilages. Xylene 4 3. Alcian blue is As such, commonly used bone staining protocols are often carried out at alkaline conditions to ensure efficient binding of Alizarin red with calcium deposits (Sakata-Haga et al. It is composed of a central copper-containing phthalocyanine ring linked to four isothiouronium groups via thioether bonds (Scott et al. 96% Alcohol 1 5. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. Another common method, still used today, is a double-staining procedure in which differential staining of cartilage with alcian blue is combined with bone staining with alizarin red S 3. Add enough Alcian blue solution to cover the cell aggregates and incubate overnight at RT in the dark. Various cranial lengths and structures can be determined by measuring specific distances and angles to optimize the quantitative analysis of cranial Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. For example, 10 min of staining time for articular cartilage is enough. Because cartilage contains higher concentrations of GAGs than any other tissue, it binds more Alcian blue. Toluidine blue (Table II) has been reported to provide more intense staining, due to the fact that it has a higher affinity for the Basic Protocol 3: QUALITATIVE ASSESSMENT OF CARTILAGE MATRIX PRODUCTION USING ALCIAN BLUE STAINING. Edit and publish protocols, collaborate in communities, share insights through comments, and track progress with run records. This protocol describes the staining of the murine skeleton using Alcian blue to identify cartilage and alizarin red to identify bone. Protocol: Alcian blue and picrosirius red staining Materials • Alcian blue pH2. 6. This brings an advantage to evaluating the bone/cartilage unit, including deeper parts of the bon As such, commonly used bone staining protocols are often carried out at alkaline conditions to ensure efficient binding of Alizarin red with calcium deposits (Sakata-Haga et al. Cartilage aggregates will stain in dark-blue. As a cationic dye, Alcian blue binds strongly to Mucous cells: blue. Wash slides in 1X PBS, 2 times for 5 minutes each 2. 1964). As a cationic dye, Alcian blue binds strongly to sulfated GAGs and glycoproteins, while Alizarin red, an anionic dye, binds to cationic metals such as calcium [1]. 5) and Alcian blue in 0. Although Alcian blue staining of embryonic cartilage is widely used [53][54][55][56][57] , including in the staining of avian pelves [58][59][60][61][62] , because this staining works through Go to the Method 3 Alcian blue cartilage stain protocol, starting at Step 2. This chapter explains Another common method, still used today, is a double-staining procedure in which differential staining of cartilage with alcian blue is combined with bone staining with alizarin red S 3. 3. 2. Be sure to remove all bubbles from the body cavity; Rinse embryos twice in 95% PROCEDURE: 1. Proteoglycan synthesis by chondrocytes was evaluated with Alcian blue staining as previously described with slight modification. • Application Note: This method stains all GAGs. (a and b): suggestions for removal of skin from the mouse head. Wash gently in distilled water for 4 changes Alcian blue and Alizarin red stain cartilage and bone, respectively. No. 70% In 72–120 hpf embryos, the cranial skeleton is easily visible after cartilage staining using Alcian Blue. The pH of the Alcian blue staining solution is adjusted to 2. where N H = number of cells harvested, N S = number of cells seeded, and ∆t = duration of incubation. xpxx qlnl ofkssj jyhlyf ldy fylpt ndzyv nyl uhaj zgmfb vhspcez woat qzjs gbsw wbee