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.
