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。
快速脱钙液 (组织病理专用) 主要由 5% 甲酸、去离子水等组成，专门用于常规病理组织的快速脱钙。本产品的特点是利用有机酸的酸性溶解钙质，脱钙速度相对较快，对组织结构有一定损伤。不同组织脱钙时间不一，一般 1-7 天即可完成。可能会破坏组织中的蛋白，如果后续需要进行抗原免疫检测，推荐使用 EDTA 脱钙。
自备材料：
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 倍体积的快速脱钙液 (组织病理专用) 中，脱钙 1~2 天或更长时间。脱钙期间应及时更换新鲜的脱钙液，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、本产品为酸性，有一定挥发性，请在通风良好处使用，并做好防护，避免吸入。
2、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。
3、厚度 5mm 的骨组织块脱钙时间一般为 0.5~2 天即可，稍大组织在 2~7 天即可。
4、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℃。
5、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。
6、脱钙用具避免使用金属容器，尽量使用玻璃容器。
7、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。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
9、该试剂仅用于科研领域，不适用于临床诊断或其他用途。
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, bone tissue and other calcium-containing tissues cannot be directly sectioned due to high hardness. Calcium must be removed to soften tissues prior to section preparation. Common decalcifying reagents include EDTA, organic acids and inorganic acids. EDTA is a mild chelating decalcifier: it binds to calcium on the outer layer of hydroxyapatite crystals to form soluble nonionic compounds, and drives the outward migration of bound calcium inside the crystals. This continuous reaction gradually dissolves hydroxyapatite crystals, and chelation can be achieved under neutral pH conditions.
Rapid Decalcification Solution (for Histopathology) is composed of 5% formic acid and deionized water, specially designed for rapid decalcification of routine pathological specimens. It dissolves calcium via organic acid acidity, delivering relatively fast decalcification yet causing certain damage to tissue structure. Decalcification duration varies by tissue type and generally takes 1 to 7 days. This reagent may degrade tissue proteins; EDTA decalcification is recommended if subsequent antigen immunoassays are required.
User-Supplied Materials
PBS buffer, distilled water, heating device or microwave oven, dedicated glass containers for decalcification, 4% paraformaldehyde fixative.
Operating Procedures (For Reference Only):
1. Do not cut bone tissue into overly thick specimens; the recommended thickness is approximately 5 mm.
2. Immerse the tissue in sufficient 4% paraformaldehyde fixative for fixation for at least 24 hours, then rinse with distilled water 3 times for 20 minutes per wash.
3. Transfer the tissue into Rapid Decalcification Solution (For Histopathology) at a volume ratio of 1:15 ~ 1:20 for decalcification over 1~2 days or longer. Replace with fresh decalcification solution regularly during treatment. The decalcification endpoint is reached when the tissue softens and can be flexed elastically and easily by hand.
To accelerate decalcification, incubate samples at 37°C. Microwave-assisted rapid decalcification is also available: set the microwave power to around 200 W and heat for 5 minutes per cycle. Repeat heating 3 to 5 times according to tissue thickness and density, with a 3~5 minute interval between cycles.
4. Rinse the tissue thoroughly with distilled water, then perform standard dehydration and embedding.
Precautions:
1. This product is acidic and volatile. Operate in a well-ventilated area with proper personal protection to prevent inhalation.
2. Decalcification time differs among tissue types. Closely monitor specimen status throughout the process to avoid over-decalcification, which damages tissues and compromises downstream experiments.
3. A 5 mm-thick bone tissue block typically finishes decalcification within 0.5~2 days, while larger specimens require 2~7 days.
4. Moderate heating speeds up decalcification; the temperature should not exceed 37~40°C. Excessively high temperature will loosen and disintegrate bone tissue, and the temperature must never go above 60°C.
5. Ensure thorough decalcification to prevent under-decalcification or over-decalcification. Optimize processing time to guarantee qualified sectioning while minimizing tissue damage from prolonged decalcification.
6. Avoid metal vessels for decalcification; glass containers are preferred.
7. Bone tissue should be fixed before decalcification, or fixation and decalcification may be carried out simultaneously. Decalcification prior to fixation is forbidden to reduce tissue damage.
8. Wear a lab coat and disposable nitrile gloves during operation for personal safety and health.
9. This reagent is limited to scientific research and shall not be used for clinical diagnosis or other uses.
Appendix:
Determination of Decalcification Endpoint (Physical Method):Puncture, pinch or clamp the tissue. Terminate decalcification once the bone tissue softens or no resistance is felt during needle puncture. This physical inspection may impair tissue architecture, so avoid excessive force or repeated testing.
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