Tms inverted phase microscope

At days 7, 14, and 21 of culture, hMSC cultures were fixed in 1.5% glutaraldehyde in sodium cacodylate (0.14 M) for 10 min. For ALP, cells were rinsed with PBS and stained with a solution of α-napthyl acid phosphate (2 mg/mL) and Fast Blue R (2 mg/mL) prepared in Tris 0.1 M at pH 10. The cultures were incubated for 1 h in the dark and washed with water at the end of the period. The presence of the staining was assessed under a Nikon TMS inverted phase microscope.

The effect of the extracts from the Mg-based substrates in the in vivo angiogenic response was evaluated by the CAM assay. Fertilized chicken embryos were incubated at 37 °C in 45% humidity for 4 days. After this period, a hole was drilled in order to expose the vascular zone of the embryo. A sterilized filter-paper disk was used as carrier for Mg extracts applied to the vascular zone. The eggs were sealed and incubated for 3 days at 37 °C. After the incubation period, the CAM was photodocumented (Nikon TMS Inverted Phase microscope).

The ability of endothelial cell cultures to self-organize the cell layer in a network of tube-like structures upon the contact with an extracellular matrix was evaluated using the Matrigel assay. Endothelial cell cultures were performed as described above. At day 4 of culture, the medium was removed and 10 mg/mL of Matrigel (BD Matrigel Matrix, BD Biosciences, Franklin Lakes, NJ, USA) was added to the culture. After an incubation of 60 min at 37 °C for the jellification of the matrix, culture medium was added to the wells and cultures kept for 24 h. Capillary tube-like formation was assessed under a microscope (Nikon TMS Inverted Phase microscope).