在病理实验中针对骨组织或一些含钙组织制作切片时，由于硬度较大不能直接制作切片，需要脱去钙质使组织变软，方可制成切片。常用脱钙试剂有 EDTA、有机酸、无机酸等，其中 EDTA 是一种较温和的螯合型脱钙剂，可与羟基磷灰石结晶的外层钙结合，形成可溶性的非离子化合物，同时又促进晶体内层的结合钙向外转移。借助这种连续性的作用使羟基磷灰石晶体逐渐融解，pH 中性时可起螯合作用。快速 EDTA 脱钙液 (免疫组化专用) 主要由 0.5M EDTA 二钠盐、磷酸盐、去离子水等配制而成，pH 值为 7.2±0.2，专门用于免疫组化的脱钙，也可用于常规组织的脱钙，但所需时间较长。相较于 EDTA 脱钙液 (10%)，本产品属于快速脱钙液；如与快速酸性脱钙液 (8%) 或 JYBL-Ⅰ 脱钙液等强酸性脱钙液相比，则本产品属于慢速脱钙液。本产品的特点是对组织结构影响最小，可以较好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、原位杂交等检测，该法脱钙速度缓慢，脱钙周期长，一般需要数周至数月，与脱钙组织大小有关。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、加热装置或微波炉、脱钙专用容器 (玻璃)、4% 多聚甲醛固定液
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、将组织置于足量的 4% 多聚甲醛固定液中固定 24 h 以上，用蒸馏水清洗 3 次，每次 20min。
3、组织转移至 15~20 倍体积的快速 EDTA 脱钙液 (免疫组化专用) 中，脱钙 10~30 天或更长时间。脱钙期间每隔 2-3 天更换新鲜的脱钙液，直到组织变软，手触碰骨组织有弹性能轻易弯折时即判断为脱钙终点。如果想加快脱钙速度，可以置于 37℃进行脱钙；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。
4、用蒸馏水冲洗数次，常规脱水、包埋。
注意事项：
1、EDTA 脱钙温和，速度缓慢，不同组织所需脱钙时间不同，脱钙期间密切观察组织情况，避免脱钙过度损伤组织影响后续实验。
2、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。
3、适当加热能加快脱钙的速度，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℃。
4、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。
5、脱钙用具避免使用金属容器，尽量使用玻璃容器。
6、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。
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。
In pathological experiments, when preparing sections of bone tissue or other calcium-containing tissues, direct sectioning is not feasible due to high tissue hardness. Calcium removal is required to soften tissues prior to sectioning. Common decalcifying reagents include EDTA, organic acids and inorganic acids. EDTA serves as a mild chelating decalcifier: it binds to calcium on the outer layer of hydroxyapatite crystals to form soluble nonionic compounds, and promotes outward migration of bound calcium inside the crystals. This continuous process gradually dissolves hydroxyapatite crystals, and chelation takes effect under neutral pH conditions.Rapid EDTA Decalcification Solution (For Immunohistochemistry) is formulated with 0.5M disodium EDTA, phosphate salts and deionized water, with a pH value of 7.2±0.2. It is specially designed for decalcification in immunohistochemistry, and can also be applied to decalcification of routine tissues with a longer processing period. Compared with 10% EDTA Decalcification Solution, this product achieves faster decalcification; however, it decalcifies more slowly than strongly acidic decalcifiers such as 8% Rapid Acid Decalcification Solution and JYBL-Ⅰ Decalcification Solution.This reagent imposes minimal impact on tissue structure and effectively preserves certain endogenous enzymes in tissues. Tissues decalcified with EDTA are eligible for subsequent tests including immunohistochemistry and in situ hybridization. This decalcification method proceeds slowly with a long cycle ranging from several weeks to months, depending on the size of tissue specimens. This product is intended for research use only and shall not be applied to clinical diagnosis or other purposes.
Required Materials to Be Prepared by User:
1. PBS buffer, distilled water, heating device or microwave oven, dedicated glass containers for decalcification, 4% paraformaldehyde fixative
Operating Procedures (For Reference Only):
1. Avoid excessive thickness when sampling bone tissue for decalcification; the recommended thickness is approximately 5 mm.
2. Immerse the tissue in sufficient 4% paraformaldehyde fixative for fixation for no less than 24 hours, then rinse with distilled water 3 times for 20 minutes each time.
3. Transfer the tissue into Rapid EDTA Decalcification Solution (For Immunohistochemistry) at a volume ratio of 1:15~1:20 for decalcification for 10 to 30 days or longer. Replace with fresh decalcification solution every 2 to 3 days during decalcification. The decalcification endpoint is confirmed when the tissue softens and can be bent easily with elasticity upon manual touch. To accelerate decalcification, incubate the sample at 37°C. Microwave-assisted rapid decalcification is also available: set the microwave power to approximately 200 W and heat for 5 minutes per cycle. Repeat heating 3 to 5 times according to tissue thickness and density, with an interval of 3 to 5 minutes between cycles.
4. Rinse the tissue several times with distilled water, followed by standard dehydration and embedding procedures.
Precautions:
1. EDTA-mediated decalcification is mild and slow, and the required duration varies among different tissue types. Closely monitor tissue status during decalcification to prevent over-decalcification which may damage tissues and interfere with downstream experiments.
2.A 5 mm-thick bone tissue block generally requires 10 to 30 days of decalcification, and most specimens finish decalcification within 14 to 60 days.
3. Moderate heating accelerates decalcification, while the temperature should not exceed 37~40°C. Excessively high temperature will cause bone tissue to loosen and disintegrate, and the temperature must never exceed 60°C.
4. Complete decalcification is necessary to avoid insufficient or excessive treatment. Optimize decalcification duration to guarantee satisfactory sectioning while minimizing tissue damage caused by prolonged decalcification.
5. Do not use metal vessels for decalcification; glass containers are strongly recommended.
6. Bone tissue should be fixed prior to decalcification, or fixation and decalcification can be performed simultaneously. Decalcification before fixation is prohibited to reduce tissue damage.
7. For personal safety and health, wear a lab coat and disposable gloves during operation.
Appendix：
Determination of Decalcification Endpoint (Physical Method):Adopt acupuncture, pinching with fingers, forceps clamping and other means. Terminate decalcification once the bone tissue softens or there is no resistance during needle puncture.Physical detection may cause certain damage to tissue structure; avoid excessive force and repeated testing as much as possible.
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