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.
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