Calcium Staining Solution (Alizarin Red S Method) - BioReagent,Biological Stain,for microscopy

Cat. No.: P1508560

QUALITÀ & PUREZZA BioReagent, Biological Stain, for Microscopy

Synonyms

Calcium Stain Kit(Alizarin Red S Method)

Storage

Room temperature

Shipped In

Normal

Panoramica

Calcium is abundant in the human body, forming the skeleton that provides structural support and playing important roles in secretion, transport, muscle contraction, and nerve conduction. Calcium exists in two forms within the body: one is ionic calcium, present in the bloodstream (referred to as blood calcium); the other is bound calcium, which combines with proteins, carbonates, or phosphates and deposits in tissues. Aside from bones and teeth, calcium normally permeates all tissues and cells and generally does not appear as a solid within tissues. However, under certain conditions, calcium may precipitate as a solid and deposit in tissues, leading to pathological calcification. The deposited calcium salts are primarily calcium phosphate, followed by calcium carbonate. Calcium salts are usually monorefringent, but calcium oxalate is birefringent. When stained with HE, calcium typically appears purplish‑blue. Many dyes can form chelates with calcium, including Alizarin Red S, purpurin, nuclear fast red, etc. Generally, these dyes are more effective in identifying moderate to large amounts of calcium than in staining trace calcium deposits lightly, but Alizarin Red S often yields more reliable results for small amounts of deposits.

Common methods for calcium salt staining include the silver nitrate method and the Alizarin Red S method. This product uses a combined staining method with Alizarin Red S and Fast Green FCF. Alizarin Red S is an anthraquinone derivative and the sodium salt of alizarinsulfonic acid, which can chelate with calcium salts in calcium carbonate or calcium phosphate to form an orange‑red complex; the combination of calcium salts and Alizarin Red produces an orange‑red precipitate, suitable for staining tissues with small amounts of calcium salts. This reagent is intended for research use only and is not suitable for clinical diagnosis or other purposes.

P1508560	Component	2×50mL	Storage
P1508560A	Alizarin Red S Staining Solution	50 mL	RT.
P1508560B	Fast Green FCF Staining Solution	50 mL	RT.

Reagents, consumables and Equipments not provided

• Fixatives: 10% neutral formalin, ethanol‑formalin, ethanol, etc.
• Graded ethanol series, distilled water, xylene or dewaxing clearing solution, neutral balsam, acetic acid aqueous solution
• Microscope

Instructions for Use (For Reference Only)

1. Routine Staining

• Fix tissue in 10% neutral formalin or ethanol, followed by routine dehydration and embedding.

• Section to 95% ethanol; place slides upright and allow to dry completely.

• Immerse sections in a staining jar containing Alizarin Red S Staining Solution for 1–5 minutes (see Note 1); rinse quickly with distilled water.

• Counterstain with Fast Green FCF Staining Solution for 1 minute; rinse with distilled water three times.

• Routine dehydration, clear with xylene or dewaxing clearing solution, and mount with neutral balsam.

2. Insoluble Calcium Staining

• Fix tissue in 10% neutral formalin fixative and dehydrate routinely, ensuring thorough dehydration in 95% ethanol.

• Embed in plastic and leave overnight at room temperature; polymerize in a 37°C oven for 2–4 days; cool in a –20°C freezer for 15–20 minutes.

• Section to a thickness of 3–5 μm; for undecalcified bone sections, remove plastic and bring to water; wash with distilled water.

• Immerse sections in Alizarin Red S Staining Solution for 5–10 minutes (see Note 2); wash with distilled water.

• Counterstain with Fast Green FCF Staining Solution for 30–60 seconds.

• Rinse quickly with 0.2–1% acetic acid aqueous solution; wash briefly under running water.

• Blot dry with filter paper; decolorize with 95% ethanol.

• Routine dehydration, clear with xylene or dewaxing clearing solution, and mount with neutral balsam.

Staining Results

Calcium deposits	Orange‑red
Background	Green

Notes

1. The staining time with Alizarin Red S should be determined based on the calcium salt content. Observe under a microscope and remove the slide for washing when calcium salts appear a deep orange‑red. Over‑staining may cause diffusion; generally, 1–2 minutes is sufficient.

2. For insoluble calcium staining, the staining time with Alizarin Red S should be determined based on the calcium salt content. Observe under a microscope until the staining reaction is strong without diffusion.

3. After staining with Alizarin Red S, calcium deposits are birefringent.

4. This method is particularly useful for identifying and detecting small amounts of calcium, such as abnormal calcification in the kidneys (hypercalciuria), which appears orange‑red.

5. Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.

Specifiche

Sinonimi

Calcium Stain Kit(Alizarin Red S Method)

Specifiche e purezza

BioReagent,Biological Stain,for microscopy

Stabilità e conservazione

Store at room temperature long term (12 months).

Condizioni di conservazione di stoccaggio

Room temperature

Spedito in

Normal

Grado

Biological Stain, BioReagent, per la microscopia

Product Line

Prodotti biologici

Application Protocols

Item-specific validated protocols

• Not specified for this item; refer to CoA/Spec Sheet and SDS.

General literature guidance (for context only; not product instructions)

• Fix samples (e.g., 4% paraformaldehyde), rinse thoroughly.
• Apply Alizarin Red S staining solution at room temperature; incubate until adequate contrast is achieved.
• Rinse extensively to remove unbound dye; document images under brightfield.
• For semi-quantitation, extract bound dye with an appropriate solvent (e.g., acidic extraction) and measure absorbance against calcium standards.

Important

• Follow your institution’s approved SOPs. Optimize timing and extraction conditions empirically for your sample type. Defer to any instructions supplied with the product lot.

Biological Roles

Applicability

• The product is a staining solution and does not possess inherent biological activity/role. It is used to visualize a physicochemical property—mineralized calcium—within biological samples.

Biochemical principle (literature)

• Alizarin Red S chelates Ca2+ ions in crystalline calcium salts (e.g., hydroxyapatite in bone-like matrix). Binding involves coordination through phenolic oxygen atoms and the anthraquinone carbonyl system, producing a colored, insoluble complex localized to mineral deposits.
• Selectivity arises from pH-dependent binding affinity and the limited solubility of the calcium–dye complex compared with complexes of other divalent cations.

Applications in biology (literature examples)

• Monitoring osteogenic differentiation by tracking extracellular matrix mineralization in cell culture.
• Visualizing calcification in vascular or soft tissues ex vivo.
• Evaluating biomineralization on scaffolds and bioceramics.

Quantitative considerations

• While primarily qualitative, AR-S staining can be adapted for semi-quantitative analysis by extracting bound dye and correlating absorbance with calcium amount via calibration curves.

Limitations (literature)

• Does not distinguish among calcium salt polymorphs (e.g., carbonate vs. phosphate) without complementary assays.
• Prior exposure to chelators (e.g., EDTA decalcification) removes calcium and abrogates staining.

Buffer Applications

Not typically applicable

• This product is a staining solution rather than a buffering system. It is not intended for use as a pH buffer in biochemical assays.

Practical note

• Because staining selectivity is pH-sensitive, users should avoid introducing strong buffers or chelators immediately pre- or post-stain unless specified by the protocol. Any buffer composition of the supplied solution is not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

Context

• The environmental footprint of calcium-staining workflows depends on solvent use, dye load, and waste management rather than stoichiometric reagents. While Alizarin Red S is a longstanding standard, greener practices can reduce impacts.

Alternatives and trade-offs (literature; not item-specific)

• Calcein (live-cell fluorescent calcium indicator): Enables live imaging without fixation; avoids acidic dye baths but requires fluorescence microscopy and may not specifically label mineralized calcium salts.
• Xylenol Orange or Arsenazo III: Alternative colorimetric chelators; some offer different pH optima or metal selectivities. Each has its own hazard/waste profiles.
• Von Kossa (silver nitrate) method: Indirectly detects phosphate via photochemical silver deposition; generates heavy-metal waste (less green) but provides crisp histological contrast.

Greener practice suggestions

• Minimize dye volume and implement microplate staining/extraction to reduce waste.
• Prefer aqueous rinses over alcohols where performance allows; consolidate solvent waste streams.
• Reuse dedicated staining dishes to limit plastic consumption; line benches to avoid repeated solvent cleaning.
• Validate shorter staining times to lower chemical usage without compromising signal.

Comparison snapshot (literature perspective)

• Alizarin Red S: Aqueous, colorimetric, fixative-compatible; waste is dye-containing water (manage as aqueous organic waste).
• Calcein: Live-cell, fluorescence-based; reduced chemical mass but requires instrumentation and phototoxicity control.

Pharmaceutical Uses

Not typically applicable

• This product is a research-grade biological stain for microscopy and is not formulated or labeled for pharmaceutical manufacturing or therapeutic use.

General context (literature)

• Colorimetric stains like Alizarin Red S may be used in non-clinical R&D settings (e.g., preclinical histology) to assess mineralization in tissues or biomaterials. They are not excipients or active ingredients in medicinal products.

Regulatory note

• For research use only (per Product Data). No pharmacopeial monograph or GMP status is implied. Users operating in regulated environments should qualify the reagent internally and consult CoA/SDS for documentation.

Physical Properties

Item-specific facts (from Product Data)

• Physical state/appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration, pH, density, refractive index, osmolality: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: Not specified for this item; refer to CoA/Spec Sheet.

General chemistry knowledge (literature/context for AR-S solutions; not item specifications)

• AR-S is typically used as an aqueous solution; common working concentrations reported in literature range from 1–2% w/v with pH adjusted around 4.0–4.3 to favor calcium lake formation. These values are examples only and not specifications of this product.
• Color: Solutions appear orange-red; staining yields red complexes with calcium salts (e.g., hydroxyapatite, calcite).

Practical implications

• pH is critical for selectivity toward calcium over other divalent cations; mildly acidic conditions are generally employed (literature). Verify the supplied pH on the CoA before use.
• If viscosity or precipitation is observed, mix gently and check expiration; do not filter unless recommended by the manufacturer, as filters may adsorb dye.

Quality and Grades

Item-specific facts (from Product Data)

• Grade/Purity: BioReagent, Biological Stain, for Microscopy.

What this means (general definitions; not item-specific specs)

• BioReagent grade indicates suitability for biological and cell culture-adjacent research workflows, emphasizing low levels of common laboratory contaminants and reliable performance in biological assays.
• “Biological Stain, for Microscopy” denotes that the formulation is intended for histology/cytology applications, emphasizing consistent chromogenic response, minimal background, and batch-to-batch reproducibility when used under typical staining protocols.

Practical quality considerations

• Lot-to-lot consistency in dye content, pH, and background is critical for quantitative calcium assays (e.g., mineralization in osteogenesis studies). Verify lot-specific CoA for concentration and pH where provided.
• Microscopy suitability implies low particulate burden to prevent artifacts; if the CoA specifies a filtration step or clarity criterion, follow accordingly.
• Documentation: For regulated labs, retain CoA and SDS with the lot number. Record any in-house qualification such as staining intensity controls (e.g., hydroxyapatite standards or previously mineralized cultures).

Stabilizers/additives

• Presence/absence of stabilizers, preservatives, or surfactants: Not specified for this item; refer to CoA/Spec Sheet.

UV/impurity limits

• Not specified for this item; refer to CoA/Spec Sheet.

Reaction and Applications

Primary application (item context)

• This is a ready-to-use Calcium Staining Solution for the Alizarin Red S method, intended for microscopy and research evaluation of calcium deposits in tissues and cell cultures (e.g., mineralized extracellular matrix during osteogenic differentiation). For research use only.

Principle (general literature)

• Alizarin Red S forms chelation complexes with Ca2+ in crystalline calcium salts (hydroxyapatite, calcite), generating insoluble red-orange dye lakes localized at mineral sites. Staining intensity correlates with calcium content, enabling qualitative visualization and, with appropriate extraction/quantification workflows, semi-quantitative analysis.

Use cases (literature examples)

• Assessment of osteogenic differentiation in MSCs or pre-osteoblasts.
• Histological detection of ectopic calcification in tissues.
• Evaluation of biomaterials/scaffolds for mineral deposition.

Practical considerations

• Fixation: Many protocols use paraformaldehyde or ethanol fixation prior to staining; fixation method influences background and dye retention.
• pH control: Selectivity for Ca2+ improves under mildly acidic conditions; verify supplied solution pH before adjusting.
• Background reduction: Thorough rinsing after staining reduces non-specific adsorption.
• Quantification (post-stain): Bound dye can be extracted (e.g., with acetic acid or CPC in literature methods) and read spectrophotometrically; ensure linearity using calcium standards.

Item-specific parameters (time, pH, concentration): Not specified for this item; refer to CoA/Spec Sheet and SDS.

Reaction Conditions

Not directly applicable as a chemical reaction reagent

• The product is used as supplied for staining rather than for driving chemical transformations.

General staining conditions (literature; not item-specific)

• Temperature: Ambient.
• Time: Commonly 5–30 minutes depending on sample type and thickness.
• pH: Often adjusted near ~4–4.3 in many AR-S methods to enhance calcium selectivity.
• Post-stain rinses: Multiple water or aqueous buffer rinses to reduce background; graded alcohol rinses may be used in some protocols prior to mounting.

Item-specific parameters (concentration, exact pH, timing)

• Not specified for this item; refer to CoA/Spec Sheet and SDS. Always validate conditions on control samples before critical experiments.

Safety and Handling

Item-specific facts (from Product Data)

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to the SDS.
• Storage: Room temperature (per Product Data). Shipped under normal conditions.

General guidance (literature/standard lab practice; not product-specific)

• PPE: Wear lab coat, safety glasses, and appropriate chemical-resistant gloves. Avoid skin and eye contact; dyes can cause persistent staining of skin/surfaces.
• Engineering controls: Use in a well-ventilated area; avoid aerosol generation. Protect benches with absorbent pads to minimize staining.
• Incompatibilities: Strong oxidizers and strong bases/acids may degrade azo/anthraquinone dyes or alter chromogenic performance. For calcium staining, avoid contact with calcium-chelating agents (EDTA, citrate) that can reduce staining efficacy.
• First aid (overview; defer to SDS): In case of eye contact, rinse with water for ≥15 min. Skin contact: wash with soap and water. Ingestion/inhalation: seek medical attention; provide SDS to responders.
• Spill/cleanup: Contain with inert absorbent; collect for appropriate disposal. Residual color may persist on porous surfaces—clean promptly.
• Waste: Dispose according to institutional and local regulations for aqueous dye-containing chemical waste.

Always consult the product-specific SDS for authoritative hazard classifications and first-aid statements.

Solvent Selection

Applicability

• This product is supplied as a staining solution; solvent selection by the user is generally not required.

General context (literature)

• Alizarin Red S staining is typically performed in aqueous media, sometimes with mild acidification. Ethanol or isopropanol rinses are often used post-stain to reduce background. Alternative formulations may include buffers to control pH, but the optimal solvent system is defined by the supplied product.

Practical tips

• Avoid introducing chelating buffers (e.g., PBS containing EDTA) immediately before staining, as they can reduce calcium availability for complexation.
• If dehydration/clearing and mounting are required for microscopy, choose mounting media compatible with dye lakes (check manufacturer notes). Aqueous mounts preserve color fidelity; nonpolar solvents can leach or alter color depending on resin.

Item-specific note

• Any required dilutions, compatible rinses, or mounting recommendations are not specified for this item; refer to CoA/Spec Sheet.

Storage and Reconstitution

Item-specific facts (from Product Data)

• Storage conditions: Room temperature.
• Shipped in: Normal.

Additional guidance (general good practice; not item-specific specs)

• Protect from light to preserve dye integrity and color performance. Store tightly capped to prevent evaporation and pH drift.
• Do not freeze unless the CoA explicitly permits; freeze–thaw can cause precipitation or color shift in dye formulations.
• If precipitate forms after storage, allow to equilibrate to room temperature and mix gently. If precipitation persists, consult the CoA or contact technical support before filtration.
• Record open date; for best reproducibility, use within the recommended shelf-life on the label/CoA (not specified here).

Reconstitution/dilution

• The product is described as a solution; any required dilutions or pH adjustments are not specified for this item; refer to CoA/Spec Sheet.

Stability indicators

• Discoloration, unexpected turbidity, or loss of staining performance can indicate degradation; compare to a control sample or prior images and replace if performance declines.

Structure and Identity

Item-specific facts (from Product Data)

• Product type: Calcium Staining Solution (Alizarin Red S Method), ready-to-use staining reagent.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• CAS, InChIKey, SMILES: Not applicable/not specified for this formulated solution.

General chemistry knowledge (for context; literature)

• The “Alizarin Red S method” commonly employs the anthraquinone dye Alizarin Red S (AR-S; typically sodium alizarin sulfonate) as the chromogenic component. AR-S complexes with calcium ions in calcified matrices, yielding an orange-red to brick-red lake.
• Structural features of AR-S (literature): an anthraquinone core bearing phenolic and sulfonate substituents; the sulfonate confers water solubility; phenolic oxygens participate in metal coordination. The 2D structure comprises a fused tricyclic ring system with keto groups at positions 9 and 10.

Notes

• As a formulated solution, the exact dye concentration, pH, and any additives/buffers are product-specific and are not provided here.

Synthetic Utility

Not applicable

• This is a formulated staining solution for microscopy, not a synthetic building block or reagent used to form new chemical bonds in preparative chemistry. For relevant information, see Reaction & Applications and Biological Roles sections addressing its use in calcium visualization.

Target Specificity

Not applicable

• Target specificity fields (antigen, epitope, species reactivity, clone, isotype) pertain to antibodies or affinity reagents. This product is a non-immunologic staining solution and does not possess epitope specificity. It complexes with inorganic calcium salts based on coordination chemistry (see Biological Roles).