Alizarin Red S is a histological stain employed to identify and localize calcium deposits within biological tissues. This technique is used to assess the presence of mineralized matrix, which is relevant in fields like toxicology and developmental biology. The stain visualizes calcification, such as in bone formation studies or in the investigation of pathological calcifications in soft tissues. Researchers use Alizarin Red S when studying bone development, tissue engineering, and mineralization processes.
Fundamental Staining Principles
The dye selectively marks calcified tissue through chelation. Alizarin Red S is an anthraquinone derivative that possesses both sulfonate and hydroxyl functional groups. These groups allow the dye molecule to bind specifically to the calcium ions (Ca²⁺) present in mineralized structures like bone or calcified cartilage.
The staining reaction requires a slightly acidic pH, maintained between 4.1 and 4.3. Operating within this narrow pH range ensures the selective formation of an insoluble precipitate, which is the calcium-Alizarin Red S complex. This complex results in a bright orange-red stain at the sites of calcium deposition. Precise control of pH is necessary for accurate and specific results, as deviations can lead to non-specific background staining.
Preparing the Tissue Sample
Successful Alizarin Red S staining requires tissue preparation that preserves calcium deposits. Tissue samples are typically fixed using a neutral buffered formalin solution, as this maintains tissue morphology and protects the mineral content. Fixation prepares the tissue for subsequent processing.
The most important consideration is the absolute avoidance of decalcification procedures. Since the stain detects calcium, any treatment that removes the mineral, such as exposure to strong acids, must be avoided. For standard histological analysis, fixed tissue is typically embedded in paraffin, and thin sections, often cut at approximately 4 micrometers, are mounted onto glass slides for hydration and staining.
Detailed Staining Procedure
The staining process requires careful preparation of the Alizarin Red S working solution to ensure optimal binding specificity. Typically, 2 grams of Alizarin Red S powder are dissolved in 100 milliliters of distilled water. This solution’s pH must then be precisely adjusted to the required range of 4.1 to 4.3 using a dilute ammonium hydroxide solution.
For paraffin-embedded tissue sections, the procedure starts with dewaxing and hydration to remove the paraffin and bring the tissue to water. Slides are immersed in xylene baths, followed by graded alcohol solutions, and finally rinsed in distilled water. Once the tissue is fully hydrated, the Alizarin Red S solution is applied directly to the slide.
Incubation time with the dye is relatively short, typically ranging from 30 seconds up to 5 minutes, with a two-minute period often yielding satisfactory results for tissue sections. The staining time is monitored microscopically to achieve a strong, but not overly diffused, orange-red color. After incubation, the excess dye is gently shaken off, and the section is carefully blotted to remove residual liquid.
The final steps involve rapid dehydration. Slides are dipped in 100% acetone, followed by a 1:1 mixture of acetone and xylene. The slides are then cleared in xylene and permanently mounted with a synthetic medium for microscopic examination.
Reading the Stained Results
The stained slides are analyzed under a standard light microscope to confirm the presence and distribution of calcium deposits. A positive result is clearly identified by the formation of an orange-red to deep red precipitate, which is sharply localized to the areas of mineralization. Background tissue remains largely colorless or very faintly stained, providing high contrast for the calcified structures.
The calcium-Alizarin Red S complex exhibits inherent birefringence. When the stained slide is viewed under a polarizing microscope, the mineralized areas appear brightly illuminated against a dark background. This technique is helpful for differentiating true crystalline calcium deposits from non-specific background staining or amorphous artifacts.