在组织切片过程中一些组织内含有骨质或钙化灶时，含钙的组织不宜直接用石蜡包埋切片，这是因为钙和石蜡之间的密度不同，较难切出完整的切片，对含钙组织最好固定之后再进行脱钙或二者同时进行，然后进行下游操作如脱水、透明、浸蜡、包埋、切片。
用于脱钙的试剂很多，脱钙剂包括有机酸、无机酸、乙二胺四乙酸 (EDTA) 以及电解法脱钙，AFIP 甲酸‑柠檬酸脱钙液主要由甲酸、枸橼酸钠等组成，其原理在于柠檬酸钠可以螯合钙离子。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 2×500mL | 储存 | | A1529146A | AFIP 甲酸溶液 | 液体 | 500mL | 室温 | | A1529146B | 柠檬酸钠溶液 | 液体 | 500mL | 室温 |
临用前，取 AFIP 甲酸溶液和柠檬酸钠溶液等量混合，即为 AFIP 甲酸‑柠檬酸脱钙液。 |
自备材料:
1、PBS、蒸馏水
2、加热装置或微波炉、脱钙专用容器 (玻璃)
操作步骤 (仅供参考):
1、骨组织脱钙时，取材不易过厚，一般大约 5 mm。
2、组织固定后，用 PBS 清洗 3 次，每次 20 min。
3、组织用蒸馏水洗清洗 3 次，每次 20 min。
4、组织转移至 20~30 倍体积的 AFIP 甲酸‑柠檬酸脱钙液中，每日更换 1 次。
5、用蒸馏水冲洗数次，常规脱水、包埋。
注意事项:
1、厚度 5 mm 的骨组织块脱钙时间一般脱钙 3~7 天即可。
2、适当加温能加快脱钙的速度，一般不应超过 37~40℃，温度过高容易使骨组织造成松散解体，尤其不可大于 60℃。
3、脱钙应彻底，防止脱钙不足或过度，脱钙程度应控制在不影响组织切片的同时尽量缩短脱钙时间，以免脱钙过长引起组织损害。
4、脱钙用具避免使用金属容器，尽量使用玻璃容器。
5、骨组织脱钙应先固定后脱钙或脱钙固定同时进行，不应先脱钙后固定，以便减少组织的损伤程度。
6、每隔一段时间检测一次脱钙程度，脱钙过度会增加组织的损伤程度，影响染色结果。
7、为了您的安全和健康，请穿实验服并戴一次性手套操作。
附录:
脱钙终点的测定 (物理法): 采用针刺、手掐、钳夹等方法，当骨组织变软或针刺时没有阻力感即可终止脱钙；物理检测法会对组织结构有一定的损害，尽量避免用力过大或反复检测。
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
| Component No. | Component Name | Appearance | 2×500mL | Storage | | A1529146A | AFIP Formic Acid Solution | Liquid | 500mL | RT | | A1529146B | Sodium Citrate Solution | Liquid | 500mL | RT |
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.
危险品化学品经营许可证（带存储）