Mc120 hd

The endothelial tube formation of human umbilical vein endothelial cells (HUVECs) was assessed for potential anti-angiogenesis properties. Briefly, 96-well plates were coated with Geltrex®, Reduced Growth Factor Basement Membrane Matrix (Invitrogen) and incubated at 37 °C for 30 min to allow gelation to occur. HUVECs were added to the top of the gel at a density of 6 × 103 cells/well in the presence of Ru-IM (1), NAMI-A, and RAPTA-C at their IC₁₀ concentrations. The diluting agent (0.1% DMSO for RAPTA-C and 0.1% water for Ru-IM and NAMI-A) served as positive control. Cells were incubated at 37 °C with 5% CO₂ for 20 h and pictures were captured with a Leica MC120 HD mounted on a Leica DMi1 microscope at 5x magnification. Quantification of tube formation was assisted by ImageJ (Fiji) angiogenesis analyzer plug-in. Tube formation quantified by number of branching points (tube nodes, TN) and total length skeleton (tube length, TL). The data were obtained from the average of three wells per treatment condition from two independent experiments.

A Leica DM500 microscope equipped with high-resolution digital camera (Leica MC120 HD) and MBF software was used for image acquisition. The number of Iba1-positive cells was determined in regions of interest (ROI) of 500 μm² (2–4 sections/mice), including the cerebellar white matter (WM), internal granule layer (IGL), Purkinje cell layer (PCL) and molecular layer (ML), both for anterior (II–III) and posterior lobules (IX–X). Microglia were also classified as ramified, hypertrophic, bushy or ameboid based on their morphologies (Martini et al., 2020 (link); Savage et al., 2019 (link)): (1) Ramified—small round soma and extended, highly branched processes; (2) Hypertrophic—larger soma and thicker, shorter and less branched processes; (3) Bushy - swollen and enlarged soma, and an excess of thicker, shorter processes with few branches; (4) Stout/Ameboid - retracted processes and irregular shape.
The quantification of Iba1-positive cells of the olfactory bulb was performed by outlining 500 μm² ROI that included the GCL. Cells and phagocytic cups were manually counted on acquired images at 20X magnification, by using the cell counter plugin from the ImageJ/Fiji NIH software (Version 1.51, National Institutes of Health).

Microscope analysis was performed with a Leica light microscope (DMRX) equipped with a digital camera (MC120 HD, Leica).