Calcium is abundant in the human body, forming bones that serve as the body's supporting framework, and also plays crucial roles in secretion, transport, muscle contraction, and nerve conduction. Calcium exists in the body in two forms: one is ionic calcium, present in the bloodstream (so-called blood calcium); the other is bound calcium, which combines with proteins, carbonate, or phosphate and deposits in tissues. Apart from bones and teeth, calcium normally permeates all tissues and cells and generally does not appear in a solid state within tissues. However, under certain pathological conditions, calcium can precipitate into solids and deposit within tissues, known as 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 H&E, calcium generally appears purplish-blue. Many dyes can chelate with calcium, including Alizarin Red S, purpurin, nuclear fast red, etc. Alizarin Red S is an anthraquinone derivative, the sodium salt of alizarinsulfonic acid, which chelates with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex. Generally, these dyes are more effective in identifying moderate to large amounts of calcium than faintly stained micro-deposits, 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 staining solution uses the modified McGee-Russell method with Alizarin Red S and Mayer's hematoxylin staining, particularly suitable for staining tissues with small amounts of calcium salts.
C774863
| Component
| 3×50mL
| Storage
|
C774863A
| Alizarin Red S Staining Solution
| 50 mL
| RT. Store in the dark.
|
C774863B
| McGee-Russell Differentiation Solution
| 50 mL
| RT. Store in the dark.
|
C774863C
| Mayer's Hematoxylin Staining Solution
| 50 mL
| 2-8℃. Store in the dark. |
Instructions for Use
I. Conventional Staining
Fix tissue in 10% neutral buffered formalin, followed by routine dehydration and embedding.
Cut sections at 5 µm thickness, and routinely deparaffinize to water.
Cover the section with Alizarin Red S Staining Solution and stain for 1~5 minutes (see Note 1). Rinse briefly with water.
Differentiate rapidly with McGee-Russell Differentiation Solution for a few seconds.
Counterstain nuclei lightly with Mayer's Hematoxylin Staining Solution for 1~2 minutes. Rinse under running tap water for 10 minutes.
Dehydrate and clear routinely, then mount with neutral balsam.
II. Insoluble Calcium Staining (e.g., for Undecalcified Bone)
Fix tissue in 10% neutral buffered formalin and dehydrate routinely. Ensure thorough dehydration in 95% ethanol.
Embed in plastic resin and polymerize overnight at room temperature, then in a 37°C oven for 2~4 days. Cool in a -20°C freezer for 15~20 minutes.
Cut sections at 3~5 µm thickness. Depolymerize undecalcified bone sections to water, then rinse with distilled water.
Immerse sections in Alizarin Red S Staining Solution for 1~10 minutes (see Note 2). Rinse with distilled water.
(Optional) Differentiate rapidly with McGee-Russell Differentiation Solution for a few seconds.
(Optional) Counterstain nuclei lightly with Mayer's Hematoxylin Staining Solution for 1~2 minutes. Rinse under running tap water for 10 minutes.
Dehydrate and clear routinely, then mount with neutral balsam.
Staining Results
Calcium Deposits
| Orange-red
|
Cell Nuclei
| Blue
|
Precautions
The staining time with Alizarin Red S should be determined based on the calcium salt content. Observe under a microscope and stop when calcium salts appear a deep orange-red, then rinse with water. Over-staining may cause diffusion; generally, 1~2 minutes is sufficient.
For insoluble calcium staining, the Alizarin Red S staining time should also be determined by calcium salt content. This method is commonly used in pathological diagnosis and research of pathological calcification and ossification, such as fracture repair, myositis ossificans, and calcification in old scar tissue.
Steps involving McGee-Russell differentiation and Mayer's hematoxylin counterstaining are not mandatory.
After staining with Alizarin Red S Staining Solution, calcium deposits are birefringent.
The Alizarin Red S method is particularly useful for identifying and detecting small amounts of calcium, such as checking for abnormal calcification in the kidney (nephrocalcinosis).
I2607488