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。
Von Ebener脱钙液主要由氯化钠、盐酸等组成，其优点是：①细胞核染色结果好；②无需水洗；其缺点是：①不宜用化学方法确定脱钙终点。
该试剂仅用于科研领域，不适用于临床诊断或其他用途。
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Von Ebener脱钙液中，脱钙3～7天或更长时间。
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, some tissues contain bone or calcified foci. Tissues containing calcium are not suitable for direct paraffin embedding and sectioning because the density differs between calcium and paraffin, making it difficult to obtain complete sections. For calcified tissues, it is best to fix the tissue first and then decalcify it, or to perform fixation and decalcification simultaneously, followed by downstream procedures such as dehydration, clearing, wax infiltration, embedding, and sectioning. Many reagents can be used for decalcification. Decalcifying agents include organic acids, inorganic acids, ethylenediaminetetraacetic acid (EDTA), and electrolytic decalcification.
Von Ebener’s Decalcifying Solution is mainly composed of sodium chloride, hydrochloric acid, etc. Its advantages are: ① good nuclear staining results; ② no washing required. Its disadvantage is: ① it is not suitable for determining the endpoint of decalcification by chemical methods.
This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Materials Required but Not Provided:
1.PBS, distilled water
2.Heating device or microwave oven, special container for decalcification (glass)
Procedure (for reference only):
1.When decalcifying bone tissue, the sample should not be too thick, generally about 5 mm.
2.After tissue fixation, wash with PBS 3 times, 20 min each time.
3.Wash the tissue with distilled water 3 times, 20 min each time.
4.Transfer the tissue to 20–30 times its volume of Von Ebener’s Decalcifying Solution and decalcify for 3–7 days or longer.
5.Rinse several times with distilled water, then perform routine dehydration and embedding.
Precautions:
1.For a bone tissue block with a thickness of 5 mm, the decalcification time is generally 3–7 days.
2.Appropriate heating can accelerate the decalcification rate, but it generally should not exceed 37–40 ℃. Excessive temperature can easily cause the bone tissue to become loose and disintegrate, and especially must not exceed 60 ℃.
3.Decalcification should be thorough to prevent insufficient or excessive decalcification. The degree of decalcification should be controlled so as not to affect tissue sectioning while shortening the decalcification time as much as possible, so as to avoid tissue damage caused by prolonged decalcification.
4.Avoid using metal containers for decalcification; use glass containers as much as possible.
5.For bone tissue decalcification, fixation should be performed before decalcification, or fixation and decalcification should be performed simultaneously. Decalcification should not be performed before fixation, so as to reduce the degree of tissue damage.
6.Check the degree of decalcification at intervals. Excessive decalcification will increase tissue damage and affect staining results.
7.For your safety and health, please wear a laboratory coat and disposable gloves during operation.
Appendix:
Determination of the decalcification endpoint (physical method): Methods such as needle puncture, manual pressing, or forceps clamping can be used. When the bone tissue becomes soft or there is no resistance upon needle puncture, decalcification can be terminated. The physical detection method will cause some damage to the tissue structure, so excessive force or repeated detection should be avoided as much as possible.
危险品化学品经营许可证（带存储）