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During tissue sectioning, when tissues contain bone or calcified lesions, calcium‑containing tissues cannot be directly embedded in paraffin for sectioning. Due to the density difference between calcium and paraffin, intact sections are difficult to obtain. For calcium‑rich tissues, decalcification should ideally be performed after fixation, or fixation and decalcification may proceed simultaneously, followed by downstream procedures including dehydration, clearing, wax infiltration, embedding and sectioning.
A variety of reagents are available for decalcification, including organic acids, inorganic acids, ethylenediaminetetraacetic acid (EDTA), and electrolytic decalcification. AFIP Formic‑Citric Acid Decalcifying Solution is mainly composed of formic acid and sodium citrate. Its working principle relies on sodium citrate chelating calcium ions. For research use only; not intended for clinical diagnosis or other clinical applications.
Contents & Storage
Mix equal volumes of AFIP Formic Acid Solution and Sodium Citrate Solution immediately before use to prepare AFIP Formic‑Citric Acid Decalcifying Solution. |
Required Materials (not supplied):
1. PBS, distilled water
2. Heating device or microwave oven, dedicated glass containers for decalcification
Procedures (for reference only):
1. For decalcification of bone tissues, specimens should not be overly thick; a thickness of approximately 5 mm is recommended.
2. After tissue fixation, rinse the samples with PBS three times, 20 min each time.
3. Rinse the tissues with distilled water three times, 20 min each time.
4. Transfer tissues into 20‑30‑fold volume of AFIP Formic‑Citric Acid Decalcifying Solution, and renew the solution once daily.
5. Rinse thoroughly with distilled water, then perform routine dehydration and embedding.
Precautions
1. For 5‑mm‑thick bone tissue blocks, decalcification generally takes 3‑7 days.
2. Moderate heating accelerates decalcification; the temperature should not exceed 37‑40 °C. Excessively high temperature may cause bone tissue disintegration, and the temperature must never go above 60 °C.
3. Achieve complete decalcification and avoid under‑decalcification or over‑decalcification. Optimize decalcification to guarantee good section quality while minimizing treatment duration, as prolonged decalcification will cause tissue damage.
4. Avoid metal containers for decalcification; glass containers are preferred.
5. Bone tissues should be decalcified after fixation, or undergo fixation and decalcification concurrently. Decalcification prior to fixation is not recommended to reduce tissue damage.
6. Check the decalcification progress periodically. Over‑decalcification aggravates tissue damage and impairs staining results.
7. Wear a lab coat and disposable gloves for personal safety and health during operation.
Appendix
Determination of Decalcification Endpoint (Physical Method)
Use needle puncture, pinching or forceps clamping. Terminate decalcification when bone tissue softens or offers no resistance to needle penetration. Physical testing may damage tissue architecture; avoid excessive force and repeated testing.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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