Phosphate buffered paraformaldehyde

Whole intact femurs were dissected from rats, fixed in phosphate-buffered paraformaldehyde (Solarbio) for 48 h, then decalcified for 2 weeks in 10% EDTA (Solarbio). Subsequently, the samples of each group were dehydrated with ethanol (Solarbio) solutions, adopting gradient concentrations of 50, 70, 80, 90, 95, and 100%. Finally, the femur tissue samples were embedded in paraffin and cut into 4-μm-thick sections using an ultra-thin microtome (Leica, Germany). The sections were stained according to the instructions of the H&E staining kit (Solarbio) and the Masson staining kit (Solarbio). Following staining, the sections were observed under the microscope and imaged.

Forty-eight hours after the transduction of U87MG and U251 cells, approximately 500 cells were cultured in each well of a 6-well plate for 2 weeks. Afterwards, cells were fixed in 4% phosphate-buffered paraformaldehyde (Solarbio, China) and then dyed with 0.1% crystal violet (Solarbio, China). ImageJ software (ImageJ, NIH) was used to determine the colony formation rate.