As for the in vivo CT systems, such as the SkyScan 1076, usually the sample is stationary so a slightly larger diameter tube could be selected to hold the specimen, but experience has indicated it is better to wait a few minutes to allow the specimen to settle before starting the CT scan so as to reduce potential motion artifacts. A high-resolution micro-CT system (SkyScan 1172, or 1173, Bruker SkyScan, Belgium) was used for assay development. HHS (See Figure 14.11.) The classic method of von Kossa (1901), which uses silver nitrate, is preferred for routine demonstration purposes on paraffin sections (Fig. Rinse in three changes of distilled water. However, scientists may only be interested in bone density measurements after they have already had some initial findings on the stained specimens; or they may not want to change their current workflow to insert a new micro-CT examination. This type of tissue also makes excellent control material. In the single-staining process, 95% ethanol was used for dehydration before KOH maceration and alizarin staining (Menegola et al., 2001, 2002). The EPA guidelines for chemicals suggest an examination of the fetal cartilage in addition to the ossified bones (Barrow, 2000). A multitude of abnormalities became apparent in these preparations, with the most striking feature being the advanced mineralization evident throughout the endochondral (arising from cartilaginous mold) skeleton (axial as well as appendicular). Alizarin Red S is used for the staining of cartilage and bone. Get the latest public health information from CDC:, Get the latest research information from NIH:, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: 2020 Jun 25;21(12):4538. doi: 10.3390/ijms21124538. 1980;108(2):202-7. doi: 10.1159/000145301. Alizarin red S method for calcium (Dahl 1952; McGee-Russell 1958; Luna 1968). Imaging Chronic Tuberculous Lesions Using Sodium [(18)F]Fluoride Positron Emission Tomography in Mice. The cells, now loosely attached to the scaffolds, are transferred with 10% (v/v) acetic acid to a 1.5-mL microcentrifuge tube with a wide-mouth pipette. For quantification of alizarin red S staining, add 800 μL 10% (v/v) acetic acid to stained scaffold and incubate at RT for 30 min with shaking. Automatic processors are available (Rousseaux, 1985 and Conway and Erb, 1995). In addition, it may be desirable to reexamine already-stained specimens using the new technology available to obtain more accurate digital 3D skeletal structure and/or ossification analysis. Calcium hydroxyapatite crystals are not visible with light microscopy (75 to 250 nm), and are nonbirefringent in polarized light. The hypothesis was that the CT imaging would be possible because both X-ray micro-CT and the alizarin red S staining are positive for calcium mineral deposition and the staining process does not seem to change the calcium in the skeletal structures (Burdan et al., 2002; Gregory et al., 2004). In order to improve the conditions of the use of the dye on biological materials, the chemical reaction between ARS and Ca2+ is studied in vitro. Karadjian M, Essers C, Tsitlakidis S, Reible B, Moghaddam A, Boccaccini AR, Westhauser F. Int J Mol Sci. This study describes a sensitive method for the recov- Whole specimens can be stained with Alizarin Red S to show the distribution of bone, especially in developing embryos. Borland SJ, Behnsen J, Ashton N, Francis SE, Brennan K, Sherratt MJ, Withers PJ, Canfield AE. The lake should be heavy but not too diffuse. 0.05% fast green FCF (CI 42053) in 0.2% acetic acid. Alizarin Red Stain kit is used for identifying calcium on tissue sections. The absence of PTHrP is therefore associated with premature maturation and ossification of the mutant cartilaginous skeleton and, hence, inappropriate acceleration of the normal endochondral ossification process. Alizarin Red S is an anthraquinone dye used to stain for calcium deposits, which are indicators of mature osteocytes. Calcium usually stains purple blue with H&E. Alizarin red is a commonly used stain to identify calcium containing osteocytes in differentiated culture of both human and rodent mesenchymal stem cells (MSCs). Ammar T. Qureshi, ... Daniel J. Hayes, in Methods in Enzymology, 2014. Alizarin Red S, Created by Global Safety Management, Inc. -Tel: 1-813-435-5161 - Control Parameters: 130-22-3, Alizarin Red S, ACGIH TLV: NA, OSHA PEL: NA Appropriate Engineering controls: Emergency eye wash fountains and safety showers should be available in the immediate vicinity of use/handling.Provide exhaust ventilation or This deformity, by causing respiratory compromise, may have contributed to the perinatal lethality of the mutants. We also scanned double-stained mouse and rat fetuses with the CT imaging method. Alizarin stained calcium deposits intensely only around pH 12. Transcutaneous electric nerve stimulation (TENS) therapy, extracorporeal shock wave therapy (ESWT),509 and a combination of phonophoresis, cross-friction massage, and range-of-motion exercises have all been reported to yield improvement.239 A more invasive treatment, arthroscopic debridement,329,669 also has produced symptomatic improvement in the treatment of calcific tendinitis. Alizarin Red S or von Kossa staining For Alizarin red S staining, VSMCs in 3 cm2 dishes were fixed in 4% formaldehyde for 10 min at room temperature, exposed to 2% Alizarin red S (Sigma) for 30 min and washed with 0.2% acetic acid. The system has a specimen carriage that is 300 mm long with an effective scannable length of 200 mm, which is suitable for most rabbit fetuses. This ability also allows us to conduct an additional micro-CT study for potentially novel data while not changing the existing workflow, since the study could be carried out after the routine evaluation. COVID-19 is an emerging, rapidly evolving situation. Do not open the tube until fully cooled.  |  These dyes include, Micro–Computed Tomography and Volumetric Imaging in Developmental Toxicology, Reproductive and Developmental Toxicology (Second Edition), Burdan et al., 2002; Gregory et al., 2004, Methods of Adipose Tissue Biology, Part B, Ammar T. Qureshi, ... Daniel J. Hayes, in, Developmental and Reproductive Toxicology, A Comprehensive Guide to Toxicology in Nonclinical Drug Development (Second Edition), Andrew C. Karaplis, Henry M. Kronenberg, in, Before skeletal evaluation can begin, the fetal carcasses that were fixed in 95% ethanol must be dehydrated, macerated, stained with, Tawnia L. Adams, ... Dennis M. Marchiori, in, Kelley and Firestein's Textbook of Rheumatology (Tenth Edition), ), and BCP crystal clumps stain with the calcium-binding dye. Alizarin red S in vivo staining - exploring optimal concentrations Proper staining of skeletal elements in fish by immersion in fluorochrome solutions demands a compromise be-tween concentration, immersion period, survival and rear-ing conditions [32]. I'm guessing that you would just have to leave it in longer.) Alizarin red S (ARS) staining has been used for decades to evaluate calcium-rich deposits by cells in culture. 10. BCP crystals may be detected as nonbirefringent globular clumps within leukocytes in some synovial and bursal fluids (see Figure 96-7), and BCP crystal clumps stain with the calcium-binding dye alizarin red S under light microscopy. All these needs will require micro-CT imaging of single- and/or double-stained fetuses. A simple histochemical technique for the identification of gunshot residue. However, it is not uncommon to discover these calcific deposits in asymptomatic regions when ordering a radiograph for another, unrelated complaint (e.g., taking a chest film, noting calcium deposits in the shoulder). In addition, in cases where the test agent is not expected to pose any reproductive or developmental issues then the embryo-fetal developmental study can be incorporated into the fertility study design to address both male and female fertility, reproductive performance, and developmental toxicity, as previously described. However, destructive neuropathic shoulder arthropathy resulting from syringomyelia or alcoholism sometimes merits consideration in the differential diagnosis of MSS (see Chapter 46). Digital maximum intensity projection volumetric visualization of an alizarin red S–stained rat fetus in a polystyrene tube holder. This ability may contribute to the generation of new data by reassessment of existing specimens. This study design is the ICH 4.1.2, which is fully described elsewhere in this chapter. It reacts with calcium, thereby helping in the diagnosis of calcium deposits. Stain the cells Carefully aspirate the destilled water and add enough Alizarin Red S staining solution to cover the cellular monolayer. Some histochemistry textbooks indicate that the alizarin red S solution, used in … Andrew C. Karaplis, Henry M. Kronenberg, in Vitamins & Hormones, 1996. The adjacent bony structures typically are not affected. The general radiographic appearance is a periarticular amorphous, cloud-like radiopacity. 14.12). Alizarin Red S is used in histology and histopathology to stain, or locate calcium deposits in tissues. The best stage for staining is after the flesh is partially cleared, and bones are visible through the surrounding tissue. Add 200 μL of 10% (v/v) ammonium hydroxide to neutralize the acid. 63.6. Scanning at 35-μm resolution covers a field of view of ∼35 mm × 17 mm in a single normal scan. From a histochemical viewpoint, the free ionic form of calcium, found in the blood, cannot be demonstrated. Hence, the absence of PTHrP influences the development of the cartilaginous skeleton prior to the initiation of endochondral ossification. Synonym: Alizarin Red S NACRES NA.75 (1958). Alizarin Red S (ARS), an anthraquinone dye, has been widely used to evaluate calcium deposits in cell culture. Dehydrate, clear, and mount in synthetic resin. 5. ARS reacts with calcium via its sulfonate and hydroxyl groups. With high-resolution CT imaging, the skeletal malformations/variations and incomplete ossifications observed exactly matched the direct observations of the stained specimens. Copyright © 2021 Elsevier B.V. or its licensors or contributors. X-ray imaging has also been used for skeletal examination of fetuses. Proper staining of skeletal elements in fish by immersion in fluorochrome solutions demands a compromise between concentration, immersion period, survival and rearing conditions [].Currently, most protocols used for vital staining of bone rely on Alizarin compounds. 20% Glycerol in 1% KOH 24hrs (swirl occasionally) 50% Glycerol in 1% KOH (swirl occasionally) 80% Glycerol in 1% KOH (swirl occasionally) 100% Glycerol (swirl occasionally) Biological Properties of Calcium Phosphate Bioactive Glass Composite Bone Substitutes: Current Experimental Evidence. Biosens Bioelectron. It is also used to study carbohydrate–boronic acid interactions. ~ 3 changes several hours each. Recommended use and restrictions on use . The skeletal phenotyping was found to be no different than that of the single stain, but the alcian blue–stained cartilage could not be clearly distinguished in the CT images in our experiments, although we were able to visualize a part of cartilaginous skeleton (data not shown). The images illustrate that the skeletal structure can be clearly visualized, based on the micro–computed tomography images. Calcium forms an alizarin red S-calcium complex in a chelation process. During assay development, it was found that special care was necessary to overcome possible motion during CT scanning since the stained specimens were pliable and imaging was conducted at the level of microns spatial resolution. Alizarin Red S (thin section staining drawer) A.R.S. Lanza D, Perna AF, Oliva A, Vanholder R, Pletinck A, Guastafierro S, Di Nunzio A, Vigorito C, Capasso G, Jankowski V, Jankowski J, Ingrosso D. PLoS One. We pH it to 4.1-4.3 with 0.5% ammonium hydroxide. It has a role as a histological dye. These dyes include alizarin red S, purpurin, naphthochrome green B, and nuclear fast red. Currently, most protocols used for vital staining of bone rely on Alizarin compounds. Use of the substance/mixture : For laboratory and manufacturing use only. Scanning and reconstruction protocols: The clear-stained rabbit fetus samples were scanned at 35-μm imaging resolution with 60 keV and 167 μA X-ray source settings to acquire 2D radiographs with a rotation step of 0.9°/180°. Potassium ferricyanide (thin section staining drawer) P.F. The size of the tubal holders was selected according to the size of the sample. (This solution can be changed daily). Xiaoyou Ying, ... Maureen H. Feuston, in Reproductive and Developmental Toxicology (Second Edition), 2017. Epub 2018 Mar 2. NSAIDs may be used, or corticosteroids may be prescribed if severe symptoms are present. None of these dyes is specific for calcium salts, although alizarin red S when used at pH 4.2 is said to be. Figure 63.6. Acute symptoms may include pain, erythema, tenderness, localized swelling or edema, and limited range of motion. No significant difference of the reconstruction protocol was applied to the clear-stained rabbit fetuses. The apparent solubility product is around 10(-7) Na+ and K+ ions do not interfere with equilibrium of the precipitation reaction but Mg2+ and soluble proteins such as bovine serum albumin react with ARS and form soluble complexes which can lead to a decrease in the ARS quantity available to react with the calcium ions. Materials Supplied by User 2015 Apr 15;66:417-22. doi: 10.1016/j.bios.2014.12.001. Again, there was a striking diminution in the cartilaginous nonstaining portions of the bones, as indicated by the diminished space between calcified–ossifìed segments. Six continuous scans were automatically conducted to cover the entire length of the rabbit fetus. Wash the cells Carefully aspirate the Alizarin Red S staining solution and wash the cell monolayer four times with 1 ml destilled water. Carol Erb, in The Laboratory Rat (Second Edition), 2006. Data on the sequence of fetal ossification in the Long-Evans strain from gestation day 14 to 21 are described by Wright et al. Summary and discussion: The hypothesis that micro-CT imaging should be able to correctly identify the skeletal structures of the single- or double-stained fetuses was confirmed; micro-CT can be used to accurately assess the stained (alizarin red S with or without alcian blue) bone elements of fetal skeletons of the mouse, rat, and rabbit. Fixation time should be brief, or ethanol can be used. Therefore a study was conducted to determine if it would be possible to develop a micro-CT assay for skeletal examination on single- and/or double-stained fetuses. Calcium salts are usually monorefringent but calcium oxalate is birefringent. 3. Chronic symptoms may persist and usually consist of mild pain and (possibly) limited range of motion. Inappropriate acceleration of the normal endochondral ossification process underlies the observed abnormalities, implicating a critical role for PTHrP in fetal skeletal development. For instance, with a high-resolution ex vivo micro-CT, e.g., the SkyScan 1172, the sample rotates during scanning, so the holder size must be matched with the specimen to restrict possible motion. Alizarin red S (ARS) staining has been used for decades to evaluate calcium-rich deposits by cells in culture. It was also possible to quantitatively measure bone microarchitecture of the stained specimens. Gross examination of these animals was unremarkable except for the skeletal deformities already described. 4. Also, if the maceration step with potassium hydroxide is carefully controlled and kept short, the cartilaginous areas of the skeleton remain partially visible as light shadowy regions (Barrow, 2000; Christian, 2001); this allows for an accurate distinction to be made between agenesis of a bone, a malformation, and a developmental delay of ossification, a variation. 2D and 3D fetal rat images: The micro-CT imaging results illustrated that the skeletal structures of the stained fetuses were clearly visible on the CT images. doi: 10.1371/journal.pone.0116468. Charles J. Churukian, in Theory and Practice of Histological Techniques (Sixth Edition), 2008. With high-resolution CT imaging, the skeletal malformations and/or variations observed were exactly matched with the direct manual observations of the clear-stained specimens (Fig. Insoluble inorganic calcium salts are a normal constituent of bones and teeth, and their demonstration is well covered in Chapter 18. In sharp contrast, no abnormalities were noted in skeletal structures that develop entirely by intramembranous ossification (without replacing a cartilaginous mold). An atlas of the fetal skeleton on gestation day 20 for the Crl: CD rat is available (Menegola et al., 2001), and Yasuda and Yuki (1996) published a comparable atlas of the skeleton of the gestation day 21 Wistar rat. Remove the Osteogenic Differentiation Medium. Churukian is in agreement with Dahl as to the higher pH and has observed that a pH as high as 7.0 produces good results. The most common forms of calcium salts occurring in these conditions are phosphates and carbonates. With different micro-CT systems, the solution may need to be different. As for the CT scan preparation, the rabbit fetus specimens were mounted in polystyrene tube holders, which were subsequently fixed to the specimen bed of the micro-CT. Ordonez AA, DeMarco VP, Klunk MH, Pokkali S, Jain SK. eCollection 2015. Alizarin Red S staining of osteoblasts (12 well plate) 1. Shake off excess dye and blot carefully. Epub 2014 Dec 2. To develop the micro-CT imaging protocols, single- (alizarin red S) or double- (plus alcian blue) stained mouse and rat fetuses were used. Stand slides on end and thoroughly air dry. The process of mesenchymal condensation to form the initial cartilaginous blastemata of long bones appeared normal. Fig. The skeletal malformations and/or variations observed (orange arrows) were exactly matched with the CT data. Clipboard, Search History, and several other advanced features are temporarily unavailable. Place sections in a Coplin jar filled with the alizarin red S solution for 5 minutes (see Notes below). Add embryo to alizarin red S/KOH solution, until bone is stained purple. 1% aqueous alizarin red S (CI 58005) adjusted to pH 4.2 or 6.3–6.5 (see Note c) with 10% ammonium hydroxide. Micro–computed tomography (CT) 3D volumetric imaging and analysis of single-stained vertebral bones of a rabbit fetus. It should be recognized that a complete assessment of developmental toxicity includes an evaluation of functional deficits that can be more appropriately performed in the F1-generation offspring naturally delivered from F0-generation animals exposed to a test agent during pregnancy. Two, four, or eight frames averaging were used to increase the signal-to-noise ratio. CPP crystals can also be detected by using alizarin red S, but they stain more weakly than BCP crystals. This method is particularly useful in the identification and detection of small amounts of calcium like those seen in heterotropic calcification in the kidney (hypercalcinosis). In addition to skeletal structure analyses, the micro-CT method makes it possible to provide quantitative 3D measurements of skeletal ossification or mineralization and bone microarchitecture on previously stained fetuses. 11. The outcome confirmed that the CT densities of the stained ossified structures were clearly measurable. In all cases, the tube should be fully filled with the same storage solution (glycerin in this case) to prevent potential specimen shrinkage during the CT imaging. These animals exhibit a multitude of skeletal deformities phenotypically consistent with a form of osteochondrodysplasia. The stained specimens can be stored indefinitely in glycerol. The method is not specific, as melanin will also reduce silver to give a black deposit. The reconstructed images were generated as 8-bit 256 gray-scale BMP images with normalized minimum and maximum values for half tone conversion. Alizarin continues to be used commercially as a red textile dye, but to a lesser extent than in the past. Suitability Certified for use in Dahl′s method for calcium These two techniques are still the primary methods for the bone and cartilage examination of fetal skeletons required in preclinical developmental toxicity studies. Polystyrene has a very low attenuation for X-rays, thereby making it an ideal sample holder. It is an organic sodium salt and an organosulfonate salt. In general, these dyes demonstrate medium to large amounts of calcium better than particulate deposits that stain weakly, the exception being alizarin red S, which tends to give more reliable results with small deposits. Description: Alizarin Red S, an anthraquinone derivative, may be used to identify calcium in tissue sections. The system employs a micro-focus X-ray source of 10 W (20–100 keV) with 5–7 μm resolution and a 10-megapixel cooled CCD sensor with optic fiber coupling to an X-ray scintillator. 1.2. Alizarin red S in vivo staining - exploring optimal concentrations. Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves. Acid dyes for lakes, which do not contain chelating groups, also stained calcium deposits selectively. (Bottom) Cross section of the wild-type (left) and mutant (right) rib cage. DI water, or nannopure, either is acceptable and the nannopure has the nice hand held dispenser for rinsing between stages 100ml plastic Nalgene bottles (3) Laboratory studies typically are unremarkable. Similar corrections to reduce the ring-artifact and beam hardening artifact were also applied. Substance name : Alizarin Red S Chemical name : Sodium 9,10-dihydro-3,4-dihydroxy-9,10-dioxoanthracene-2-sulfonate monohydrate CAS-No. The fetal specimens were previously eviscerated and skinned, and washed in tap water. A solution of 75% ethyl alcohol (ETOH) and alizarin red S can be substituted for KOH-alizarin red S to stain bone. Subject: Re: [Histonet] Alizarin Red S Staining Protocol We always used a 2% Alizarin red S (never tried it with 1%. Alizarin Red S Solution preparation: Dissolve 2 g Alizarin Red S (Sigma, Cat# A5533) in 100 ml of ddH 2 O and adjust pH to 4.1-4.3 with HCl or NH 4 OH. It is commonly used to stain embryo skeletons in cleared whole mounts, usually of small mammals. For precipitation to occur a neutral pH (4 less than pH less than 8) is required; the stoichiometric ratio is 1:1. In the homozygous mutants there was a striking paucity of the normally unstained cartilaginous portions of the ribs and sternum (Fig.  |  The use of various dyes that act by forming chelate complexes with calcium has been practiced by many workers. The staining time is dependent on the amount of calcium present. In summary, mice homozygous for the mutated PTHrP gene die at birth, perhaps of asphyxia. Relative paucity of osteophytes (so-called atrophic degenerative arthritis) on plain radiographs and the sizeable glenohumeral joint effusions with abundant synovial fluid BCP crystalline material associated with MSS help distinguish MSS from primary OA of the glenohumeral joint. After staining, the ossified skeleton can be examined and checked for the presence, size, and shape of every bone (Taylor, 1986; Barrow, 1990; Christian, 2001). Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions. Alizarin red S (ARS) is used for histological characterization of calcium. However, differences in the length of long bones and associated deformities were apparent by day 14.5 of gestation. HADD is characterized by basic calcium phosphate crystals in the periarticular soft tissues, especially the tendons (calcific tendinitis). This method may be slightly more sensitive than the von Kossa stain for calcium. 2019 Jan 14;20(2):305. doi: 10.3390/ijms20020305. Alizarin Red is used in a biochemical assay to determine, quantitatively by colorimetry, the presence of calcific deposition by cells of an osteogenic lineage. Calcium deposits are birefringent after staining with alizarin red S. McGee-Russell recommends using the alizarin red S at pH 4.2.
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