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。EDTA 是一种相对较好的螯合脱钙剂，对组织结构影响最小，可以较好的保存组织的某些酶类，经 EDTA 脱钙后的组织可以进行免疫组化和原位杂交染色，但是该法脱钙速度太慢，一般脱需要数周至数月。
本品主要由 EDTA、碱等组成，脱钙以后可以用于电镜方面；其优点是：①作用时间中等；②对组织的结构损害小；③脱钙完全。该脱钙液特别适用于日常病理科骨组织标本的脱钙，但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、蒸馏水或 PBS
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 2mm。
2、组织固定于预冷的固定液 12～24h 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 倍体积的电镜脱钙液中，每日检查脱钙的情况，直至骨片完全脱钙为止；一般密质骨约 3~4 周，松质骨 7~10 天。
5、如果想加快脱钙速度，可以置于 37℃ 进行脱钙。
6、用蒸馏水冲洗数次，脱水、包埋。
注意事项：
1、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℃。
2、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。
3、脱钙用具避免使用金属容器，尽量使用玻璃容器。
4、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。
5、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。
6、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。
When tissues contain bone or calcified lesions during sectioning, calcium-containing specimens cannot be directly embedded in paraffin for slicing. Due to the density difference between calcium and paraffin, it is difficult to obtain intact sections.For calcium-containing tissues, decalcification is recommended after fixation, or fixation and decalcification can be performed simultaneously, followed by subsequent procedures including dehydration, clearing, wax infiltration, embedding and sectioning.There are various decalcifying reagents, including organic acids, inorganic acids, ethylenediaminetetraacetic acid (EDTA) and electrolytic decalcification methods.EDTA serves as a superior chelating decalcifier with minimal impact on tissue structure and excellent preservation of certain enzymes in tissues. Tissues decalcified with EDTA are compatible with immunohistochemistry and in situ hybridization staining. However, this method features an extremely slow decalcification rate, which usually takes several weeks to months.
This product is mainly composed of EDTA, alkali and other components, and the treated samples can be applied to electron microscopy observation.Advantages: ①Moderate reaction duration; ②Slight damage to tissue structure; ③Complete decalcification.This decalcifying solution is especially suitable for routine decalcification of bone tissue specimens in pathology departments. Chemical methods are not recommended to judge the end point of decalcification.This reagent is only for scientific research purposes and must not be used for clinical diagnosis or other applications.
Materials to Be Prepared by Users:
1. Distilled water or PBS
2. Heating device or microwave oven, dedicated glass container for decalcification
Operating Procedures (For Reference Only):
1. The thickness of sampled bone tissue shall not be excessive, with a recommended thickness of approximately 2 mm.
2. After the tissue is fixed in pre-cooled fixative for 12～24 hours, rinse it with PBS 3 times for 20 minutes each time.
3. Rinse the tissue with distilled water 3 times for 20 minutes each time.
4. Transfer the tissue into Electron Microscope Decalcifying Solution with a volume 20 to 30 times that of the sample. Check the decalcification progress daily until the bone fragment is fully decalcified.
Generally, compact bone requires about 3～4 weeks, while cancellous bone takes 7～10 days.
5. Decalcification at 37℃ is allowed to accelerate the reaction if needed.
6. Rinse the sample several times with distilled water, then perform dehydration and embedding.
Precautions:
1. Appropriate heating can speed up decalcification. The temperature shall not exceed 37～40℃; excessively high temperature may cause loosening and disintegration of bone tissue, and the temperature must never be higher than 60℃.
2. Ensure thorough decalcification to avoid under-decalcification or over-decalcification. Control the decalcification degree to guarantee qualified sectioning while shortening the treatment time as much as possible to prevent tissue damage caused by prolonged decalcification.
3. Metal containers are forbidden for decalcification; glass containers are preferred.
4. Bone tissue shall be fixed before decalcification, or fixation and decalcification shall be carried out concurrently. Decalcification prior to fixation is prohibited to minimize tissue damage.
5. Inspect the decalcification status regularly. Over-decalcification will aggravate tissue damage and interfere with staining outcomes.
6. Wear a lab coat and disposable gloves during operation for personal safety and health.
Appendix:
Determination of Decalcification Endpoint (Physical Method): Adopt acupuncture, pinching or clamping methods. Terminate decalcification when the bone tissue softens or no resistance is felt during needle penetration.Physical detection may cause certain damage to tissue structure; avoid excessive force or repeated testing.
ZJ26F0939290
危险品化学品经营许可证（带存储）