An Optimized Method For Embedding Multiple Undecalcified Mice Tibias In Plastic Block

Objective To optimize the method for embedding multiple undecalcified mice tibias in one plastic block and to explore the method for reducing the detachment rate of plastic slides during sectioning and de-plastic and hydrationMethods 30 undecalcified tibias from 15 B6 mice were used for this experiment.Injected twice osteocalcin(2.5mg/ml)to each mouse with interval time of 72 hours Sacrificed all the mice 2 days after second injection by carbon dioxide.Took 30 tibias from all the mice and used 10%formalin to fix them.Used serial increasing concentration ethanol for tibias dehydration.Embedded the tibias as cylindrical shape with a diameter of 4 mm and then cut off each bone at the position of 1/4 distance to the proximal tibia.The 3/4 part of those tibias were taken for re-embedding with the section planes standing flatly by sticking the cross surface onto a flat plate and 5 bones per block.Samples were randomized into control and experimental groups when 3 processes were introduced before embedding,sectioning and staining respectively,including ①flowing carbon dioxide(CO2)into embedding solution;②paste with embedding solution onto section surface;③heat the slides under 95℃ for 15 min.Polymerization time,slide detachment rate,bone formation and osteoblast parameters were analyzed latterlyResults 5 tibias per block and 6 blocks were made and all the cross section of the tibias were 1/4 distance to the proximal tibia.All the blocks were completely polymerized and suitable for section.Flow CO2 into embedding solution reduced polymerization time and increased complete polymerization rate.CO2 group’s blocks were completely polymerized 4 hours after making embedding solution(n=10).While 7 of the control group’s blocks were completely polymerized and 3 of them were not.Paste embedding solution significantly reduced detachment rate(p<0.001)but not changed bone formation analysis(p>0.05).The average detachment rate of the Paste group slides wasl 1.25%±0.06 while that of the control group was 67.5%±0.12.Heated slide showed significantly low detachment rate(p<0.01)but not changed osteoblast analysis(p>0.05).The average detachment rate of the Heated group slides was28.97%±0.29 while that of the control group was 56.60%±0.22Conclusion To embed multiple undecalcified mice tibias in the same block by 2-time embedding works well.It makes that to section multiple tibias at one time possible.To flow carbon dioxide into the embedding solution can reduce the polymerization time and increase the complete polymerization rate.To paste the embedding solution can reduce the detachment rate of slides during sectioning bur not affect the observation and analysis of osteocalcin labeling.To heat the slide can reduce the detachment rate during de-plastic and hydration but not affect the staining of toluidine blue and analysis.It may be an ideal method to analyze the histology of undecalcified bones.