小鼠胫骨不脱钙树脂包埋法的改良与应用

目的改良树脂包埋复数小鼠不脱钙胫骨的方法，提高树脂包埋法的效率及稳定性，探索减少树脂切片脱片的方法。方法取15只B6小鼠共30胫骨，标记、固定、脱水、渗透后将其包埋于直径4 mm的圆柱型树脂块中，胫骨近端1/4处切断后再对剩余的3/4胫骨包埋。在包埋时将5个胫骨切面贴于平整树脂板并包埋于同一树脂块中。分别在包埋液制备、切片、染色3个阶段随机将样本分为对照组及实验组，分别在3个改良组中采用以下方案处理，包括：向包埋液冲入CO2；在切面上涂抹包埋液；在脱塑水化前以95 ℃加热切片15 min。分析树脂凝固时间、切片脱片率及骨形成、成骨细胞定量指标。结果成功包埋6个树脂块，每个树脂块内有5个胫骨，胫骨横截面于同一平面上，树脂块凝固完全，适于切片。向包埋液冲入CO2可以缩短树脂块凝固时间，提高凝固成功率。在切面上涂抹包埋液可以显著降低切片时的脱片率（P<0.05）且不影响骨标记的荧光分析（P>0.05）。在脱塑前加热切片可以显著降低脱塑水化后的脱片率（P<0.05），且不影响成骨细胞定量分析（P>0.05）。结论复数小鼠不脱钙胫骨的树脂包埋改良法有效可行，这可能是一种理想的不脱钙骨组织研究方法。

An optimized method for embedding undecalcified mouse tibias in plastic blocks

Objective To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve theefficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. Methods Thirty undecalcifiedtibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibiaswere embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and theremaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on thesurface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding,sectioning and staining. In the 3 groups with modified embedment, flowing CO2 was added into the embedding solution,embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerizationtime, slide detachment rate, bone formation and osteoblast parameters were analyzed. Results We prepared 6 plastic blocks,each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitablefor sectioning. Flowing CO2 into the embedding solution reduced the polymerization time and increased the rate of completepolymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of thesections (P<0.05) without affecting bone formation analysis (P>0.05). Heating the slides significantly lowered the detachmentrate of the sections (P<0.05) without affecting osteoblast analysis (P>0.05). Conclusion The optimized method allows effectiveembedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis ofundecalcified bones.