Ultrathin (~70 80 nm) oriented serial sections were obtained with a UC6 ultramicrotome (Leica Microsystems), collected on uncoated 300-mesh copper grids (TAAB Laboratories), stained with saturated uranylacetate and lead citrate, and imaged by TEM.
Longitudinal labio- (bucco-) lingually oriented ground sections were prepared with a diamond blade microtome (Leica 1600) following the protocol of Caropreso et al. [37].
Transversely oriented sections of the frozen material were processed for routine histochemical analysis.
On favourably oriented sections, head-to-head vessel sprouts are recognizable at various steps of the process growth journey, while they grow to fill the gap between their respective parental vessels, and finally fuse head-to-head to form a new, patent, anastomotic collateral (Fig. 2A C) (see also Online Resource 1).
5 µm oriented sections crossing inferior pole, optic nerve and superior pole were cut and stained with toluidin blue.
For the 2.5 mm beads, detection decreased to 78-94% for 5 mm collimation/pitch 2 scans when the phantom sections were oriented parallel to the gantry (p = 0.01).
At the age of 2 and 9 weeks of age, mice were sacrificed and pancreata dissected, weighed and fixed in formalin followed by paraffin embedding, orienting pancreata such that sections were cut along the head-tail axis.
paraformaldehyde at 4°C under continuous shaking followed by paraffin embedding, orienting pancreases such that sections were cut along the head tail axis.
After TS-2, a transversely oriented tissue section is taken from the caudal mesencephalic stump, either at the intercollicular level or the level of the rostral colliculi (TS-2′).
Frozen samples were then oriented and 6 µm thick cryostat sections were cut for immunohistochemical staining and analysis using light microscopy, as previously described (Di Stefano et al, 2002).
