Thermo microm hm355s

Both decalcified and undecalcified histological analyses were conducted on the defect zone samples to investigate cellular and structural changes underlying healing. After euthanasia, defect zones were collected and freed from soft tissue. The samples were fixed in 4% paraformaldehyde (PFA) and then decalcified using 4% PFA and 14% ethylenediaminetetraacetic acid (EDTA) for 8 weeks at 4 °C. After embedding, paraffin blocks were sectioned into 5 µm thick slices using motorized rotary microtome (Thermo/Microm HM355S, Thermo Scientific GmbH, Karlsruhe, Germany), whereas the fluorochrome labelled samples were embedded in PMMA medium.
Decalcified sections were used for toluidine blue staining [27 (link),28 (link),29 (link)] to carry out the qualitative analysis of the defect zone across time points.

Left femora were harvested with surrounding muscle tissue after euthanasia and fixed in 4% paraformaldehyde (PFA) for the histological examinations as described previously [52 (link),89 (link)]. The bone samples for histology were collected at D7, D10, D14, D21, and D28. After 48 h of fixation at 4 °C, the decalcification of bone samples was carried out using a 1:2 solution of 4% PFA and 14% Ethylenediaminetetraacetic acid (EDTA) at 4 °C for 3 weeks. The solution was changed every three days. The bone samples were embedded in paraffin post-dehydration and stored at 4 °C for 24 h before sectioning. The blocks were cut into 5 µm thick sections using a motorized rotary microtome (Thermo/Microm HM 355S, Thermo Scientific GmbH, Karlsruhe, Germany).