Objective:It is an interesting challenge to study green fluorescence protein(GFP) labled cells in osseous stuff(including bone bioscaffolds and nature bone), for osseous stuff is very hard and with poor lucency. To solve this problem, the authors tried to establish a semi-solid decalcification system. Methods:①Primary Mesenchyma stem cells were separated from rabbit bone marrow, modified with recombined lentivirus with GFP gene(BMSC-GFP). ②BMSC-GFP were inoculated in bone bio-scaffold of β-tricalcium phosphate(β-TCP); bio-scaffold with cells were fixed with 4% paraformaldehyde phosphate buffered saline(PBS). ③Different agents for decalcification were tested, after that agents selected were added into semi-solid system to find proper combination. ④Frozen sections were made after decalcification in semi-solid system for 1~4w, paraffin sections and plastic embedding sections were used as controls. Results:①GFP+ cells can be found growing on internal side of the holes of bio-scaffold by fluorescence invert microscope. ②BMSC-GFP cells remain at the site where they grow during decalcification in semi-solid system, discovered by fluorescence invert microscope. ③Comparing with plastic embedding sections, semi-solid system and frozen sections can keep the fluorescence and original modality, however, plastic embedding section can only be applied when scaffold is very small, while paraffin sections do not work to scaffold. Conclusion:With the own-made semi-solid decalcification system, GFP+ cells growing inside bone bio-scaffolds can keep fluorescence, modality in situ. It will be great help to the study of mechanism in many fields such as bone and cartilage tissue engineering, bone marrow transplantation and so on.