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。
柠檬酸盐脱钙液是缓冲脱钙液，属于缓慢脱钙液，主要由柠檬酸、柠檬酸盐、硫酸锌等组成，pH 值约为 4.5；其优点是：①组织染色结果好；②对组织的结构损害小；
其缺点是：①脱钙速度很慢，不适合常规标本脱钙使用；②脱钙后组织会稍微变硬。该试剂仅用于科研领域，不宜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 5mm。
2、组织固定后，用 PBS 清洗 3 次，每次 20min。
3、组织用蒸馏水清洗 3 次，每次 20min。
4、组织转移至 20~30 倍体积的柠檬酸盐脱钙液中脱钙 15~30 天或更长时间；如果想加快脱钙速度，可以置于 37℃进行脱钙；如果必要，更换新的柠檬酸盐脱钙液继续脱钙，多数组织脱钙 2 周～3 个月即可，每周更换一次直至终点；亦可采用微波快速脱钙法：微波炉设在 200W 左右的档位，每次加热 5min，依据组织厚度和密度重复 3~5min，中间间隔 3~5min。
5、用蒸馏水冲洗数次。
6、常规脱水、包埋。
注意事项：
1、厚度 5mm 的骨组织块脱钙时间一般脱钙 10~30 天即可，大多数在 14~60 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, tissues containing bone or calcified foci are not suitable for direct paraffin embedding and sectioning. This is because the density difference between calcium and paraffin makes it difficult to obtain intact sections. For calcium-containing tissues, it is best to perform decalcification after fixation or carry out fixation and decalcification simultaneously, followed by downstream procedures such as dehydration, clearing, wax infiltration, embedding and sectioning. There are many reagents available for decalcification, including organic acids, inorganic acids, ethylenediaminetetraacetic acid (EDTA) and electrolytic decalcification methods.Citrate Decalcification Solution is a buffered decalcifying solution and belongs to the category of slow decalcifying solutions. It is mainly composed of citric acid, citrate salts, zinc sulfate and other components, with a pH value of approximately 4.5. Its advantages are as follows: ① It yields excellent tissue staining results; ② It causes minimal damage to tissue structure. Its disadvantages include: ① The decalcification speed is very slow, making it unsuitable for decalcification of routine specimens; ② Tissues will harden slightly after decalcification. This reagent is only for scientific research use and is not suitable for clinical diagnosis or other purposes.
Required Materials to Be Prepared by the User:
1. PBS, distilled water
2. Heating device or microwave oven, dedicated glass containers for decalcification
Operating Procedures (For Reference Only):
1. When decalcifying bone tissue, do not take overly thick samples; the recommended thickness is approximately 5 mm.
2. After tissue fixation, rinse the tissue with PBS 3 times, 20 minutes each time.
3. Wash the tissue with distilled water 3 times, 20 minutes each time.
4. Transfer the tissue into 20~30 times its volume of Citrate Decalcification Solution for decalcification for 15~30 days or longer. If you want to accelerate the decalcification speed, you can place the tissue at 37°C for decalcification. If necessary, replace with fresh Citrate Decalcification Solution to continue decalcification. Most tissues can be completely decalcified within 2 weeks to 3 months, and the solution should be replaced once a week until the decalcification endpoint is reached. Microwave-assisted rapid decalcification is also available: set the microwave to about 200W, heat for 5 minutes each time, repeat the heating for 3~5 minutes according to the thickness and density of the tissue, with an interval of 3~5 minutes between each heating cycle.
5. Rinse the tissue several times with distilled water.
6. Perform routine dehydration and embedding.
Precautions:
1. For a 5 mm-thick bone tissue block, the decalcification time is generally 10~30 days, and most of them can be completely decalcified within 14~60 days.
2. Moderate heating can accelerate the decalcification speed, but the temperature should not exceed 37~40°C. Excessively high temperature is likely to cause bone tissue to loosen and disintegrate, and the temperature must never exceed 60°C.
3. Decalcification should be thorough to prevent insufficient or excessive decalcification. The degree of decalcification should be controlled to ensure that it does not 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; glass containers are preferred.
5. For bone tissue decalcification, fixation should be performed first followed by decalcification, or fixation and decalcification can be carried out simultaneously. Decalcification before fixation is not allowed to reduce tissue damage.
6. Check the degree of decalcification at regular intervals. Excessive decalcification will increase tissue damage and affect staining results.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
Appendix:
Determination of Decalcification Endpoint (Physical Method): Use methods such as needle puncture, manual pinching, and forceps clamping. Terminate decalcification when the bone tissue softens or no resistance is felt during needle puncture. The physical detection method may cause certain damage to the tissue structure, so avoid using excessive force or repeated detection.
ZJ26F0938905
危险品化学品经营许可证（带存储）