sign in sign up
<<
Home , Dye, Lake pigment

Research Article

Alizarin S – A procedure for bones S. Kameswari, S. Sangeetha, Karthik Ganesh Mohanraj*

ABSTRACT

Aim: This study is to know about the skeletal framework of fish by staining the bones using red S. This is widely used to evaluate the staining bones in cell culture, for fabrics. It became the first natural to be made synthetically. Alizarin red is the main constituents for the manufacture of the madder lake . Materials and Methods: The chemicals required for the procedure are Formaldehyde solution, 50% hydroxide (KOH), 1% , Glycerol and Distilled . The fish were killed and dipped in water with neutral formalin. Then, it was washed to remove all sludge. The scales were removed and immersed in solution containing 1% of Alizarin red S and 50% of KOH and left undisturbed. Results: This result in the staining of bones in pinkish- color which helps to know about the skeletal framework. Conclusion: This in vitro study helps to know about the skeletal framework of fish through the stain Alizarin red S. Thus, the staining of bones helps in disease detection and deposition of calcium. KEY WORDS: Disease detection, Formalin fixed tissues, Skeletal framework, Staining

INTRODUCTION substances on advancement of bones and ligaments so as to record the dimension of plausible disfigurement. Alizarin red S is widely used to evaluate the staining Staining of skeletal framework in laboratory is an bones in cell culture. It has been used for the past several important step in dermatological investigations. centuries as an important red dye, for dyeing textile Alizarin red as a color indicator for skeletal bony fabrics.[1] It became the first to be made parts has a high affinity for binding with calcium synthetically. Alizarin red is the main constituents for and has been used for many years to stain bones. the manufacture of the madder . In the past Furthermore, staining with Alizarin red which is 10 years, the use of has been existed followed by potassium hydroxide (KOH) clearing for the investigation of human bone tissues. Thus, for has been used for many years as a simple and reliable the perception of the bone structure, alizarin recoloring technique.[2-5] The scale reagent, a -based agent [8] strategy as utilized by Potthoff, 1984, and adapted can also be used to make soft tissue transparent,[6-7] by S. Helland, Nofima Marin, was utilized with some has used in bone staining with Alizarin red S. alternation to the stain the entire fish so as to watch the bone structure. The recoloring method will help in After that, all the 2-fold skeletal recoloring techniques the bone investigation. A relative, developing method have been finished by Alizarin red as bone and for staining the skeleton in transparent specimen is ligament markers. Distinctive conventions have developed using Alizarin red S stain. A simple, quick been proposed since 1897 for recoloring skeletons diagnosing method using Alizarin red S and ordinary and ligaments. Every one of them had its claim light microscopy to detect bone framework plays a confinements. In many studies, an equivalent major role in nowadays. convention was utilized in research center creatures with various ages. Notwithstanding, this investigation Skeletal recoloring is an approach to contemplate attempted to clarify the distinction of recoloring in the impact of numerous compound or homegrown grown-up and incipient organism of research facility. Alizarin staining has been in use for more years which Access this article online is also used to assess calcium deposit[9-10] by cells in culture. This staining technique may have various Website: jprsolutions.info ISSN: 0975-7619 applications in the field of industry, pharmaceutical,

Department of Anatomy, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu,

*Corresponding author: Karthik Ganesh Mohanraj, Department of Anatomy, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India. Phone: +91-9944953239. E-mail: [email protected]

Received on: 17-12-2018; Revised on: 05-01-2019; Accepted on: 16-02-2019

890 Drug Invention Today | Vol 12 • Issue 5 • 2019 S. Kameswari, et al. and in environmental use.[11] It is known to stain more recoloring arrangement was used. Two fishes were commonly the fetal bones rather than the cartilaginous taken in which one was dissected and the other was part of the skeleton which may lead to the failure to without dissection. The stain was left undisturbed. indicate some of the vital qualitative alternation in While observing on the 1st day, the bone was stained skeletal morphology. a little bit and the 2nd day, the bone was observed with more . Like this, it was left for about 5 days. MATERIALS AND METHODS Preservation The fresh fish specimen was sacrificed and fixed in Depending on the degree of staining, the specimen 40% formalin. Then, it was cleared in gradations of was noticed regularly. Final preservation was made in KOH. The staining agent Alizarin red S was used to the glass bottle containing 600 ml of fresh water with stain the skeletal framework after removing scales. 99.5% of glycerol with rubber stopper. The fish Indian mackerel (Rastrelliger kanagurta) which would be approximately 84 cm and 18 cm and RESULTS weight 2.3 kg was taken for the study. Finally, the bone was stained completely on the The chemicals that were used for the staining 5th day which showed a pinkish-violet color. Staining procedures are as follows: of bones using Alizarin red S contributes to a strong • Formaldehyde solution color differentiation of the bone tissues. The contrast • KOH – 50% in color is the best seen on bone macrosections. This • Alizarin red S – 1% method of surface staining of bones tissues with • Glycerol Alizarin red follows from the verified methods of • Water. Johnson, Williams, etc. Killing DISCUSSION The fish were killed and it was then dipped under normal running water for a couple of minutes. After, it It is very much used in disease detection and also in was made clear that it was dead completely. medical studies involving calcium. The important thing is that it is also used in fetal staining of bones. The Fixation alizarin coloring agent is bound to the surface of the The fish were then dipped in 2 L of tap water with bone only, and the complete clarification of tissues is not [8] 10% of neutral formalin for over 48 h. The fish were possible. In case of larger size fish, it was put forward left undisturbed during the fixation. It was noticed to steam the fish first, facilitating removal of scales, each and every 5 h of a day. skin, etc. Another way to notice the bone structure is by X-ray, but it is costly similar to staining method. This Washing method technique would be helpful in the osteological Before continuing with the recoloring technique, that studies of fishes. Our highly transparent bone staining is after the fixation, the fish were washed completely specimens will pass the clear picture of [12] under running faucet water for somewhere around 1 h the skeletal system to be obtained. to expel abundance sludge and formalin. Then, the fish This bone staining procedure is achieved[13] to be were made free from all the sludge and formalin which developed to the multistaining system for the bone would help the scaling process to be done quickly. and different structure by added with immunostaining Scaling or genetic labeling procedure. However, whole-mount bone staining using the RAP system is possible to Once the washing was over, fish were completely free apply to larger specimens.[14,15] from scales which help in the staining of bones more easily. The scaling process was done for about 15 min CONCLUSION and all scales were completely removed. This in vitro study helps to know about the skeletal Bone Staining framework of fish through the stain Alizarin red S. Recoloring specialists combine with tissues and Thus, the staining of bones helps in disease detection enhance the perceivability of their segments by giving and deposition of calcium. This staining process is shading to them. The fish were then set in a glass more success in young organisms as the bones are container containing 5% of KOH. There recoloring very transparent in it. This staining of bones could arrangement containing 1% Alizarin red S was added also be done in fetus and in bone investigation. The drop to the naturally arranged 5% KOH arrangement alizarin coloring agent is bound to the surface of the holding the examples till the medium (KOH) bone only, and the complete clarification of tissues is indicated pinkish-violet shading. Around 15 ml of the not possible.

Drug Invention Today | Vol 12 • Issue 5 • 2019 891 S. Kameswari, et al.

REFERENCES of Ichthyology and Herpetology Special Publications No. 1. Lawrence, USA: Allen Press, Inc.; 1984. p. 35-7. 1. Pirillo S, Ferreria ML, Rueda EH. The effect of pH in the 9. Horobin RW. How do impart color to different component adsorption of alizarin and eriochrome R onto iron of the tissues? In: Kumar GL, editor. Educational Guide Special oxides. J Hazard Mater 2009;168:168-78. Stains and H and E. Carpinteria, California: Dako; 2010. 2. Mall FP. On ossification centers in human embryos less than p. 159-66. one hundred days old. Am J Anat 1906;5:433-58. 10. Adkins KF. Alizarin red S as an intravital fluorochrome in 3. Dawson AB. A note on the staining of the skeleton of cleared mineralization tissues. Stain Technol 1965;40:69-70. specimens with alizarin red S. Biotech Histochem 1926;1:123‑4. 11. Susaki EA, Tainaka K, Perrin D, Kishino F, Tawara T, 4. Staples R, Schnell VL. Refinements in rapid clearing technic in Watanabe TM, et al. Whole-brain imaging with single-cell the KOH-alizarin red S method for fetal bone. Stain Technol 1964; resolution using chemical cocktails and computational analysis. 39:61-3. Cell 2014;157:726-39. 5. Simon EV, van Horn JR. A new procedure for whole-mount 12. MC. A rapid method for cleaning and staining specimen alican blue staining of the cartilaginous skeleton of chicken for the demonstration of bone. Ohaio J Sci 1952;52:31-3. embryo, adapted to the the cleaning procedure in potassium 13. Wassersug RJ. A procedure for differential staining of cartilage hydroxide. Acta Morphol Neerl Scand 1971;8:281-92. and bone in whole formalin-fixed vertebrate. Stain Technol 6. Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, 1976;51:131-4. Noda H, et al. Scale: A chemical approach for fluorescence 14. Park EH, Kim DS. A procedure for staining cartilage and bone imaging and reconstruction of transparent mouse brain. Nat of vertebra larvae while rendering all other tissue transparent. Neurosci 2011;14:1481-8. Stain Technol 1984;59:269-72. 7. Abe G, Lee SH, Chang M, Liu SC, Tsai HY, Ota KG. The origin 15. Song J, Parenti LR. Clearing and staining whole fish specimens of the bifurcated axial skeletal system in the twin-tail goldfish. for simultaneous demonstration of bone, cartilage, and nerves. Nat Common 2014;5:3360. Copeia 1995;1:114-8. 8. Potthoff T. Cleaning and staining technique. In: Moser HG, Richards WJ, Cohen DM, Fahay MP, Kendall AW Jr., Richard SL, Source of support: Nil; Conflict of interest: None Declared editors. Ontogeny and Systematics of Fishes. American Society

892 Drug Invention Today | Vol 12 • Issue 5 • 2019