Eclipse te2000 s fluorescent microscope

Immunofluorescence was used to determine blood vessel density with CD31 (Abcam), a marker for vascular endothelium, and the blood vessel staining was analyzed with a Nikon Eclipse TE2000-S fluorescent microscope (magnification, 100X) using the 488-excitation wavelength. The vessel density was determined by counting the number of blood vessels in 10 fields (magnification, 100X) of slides from each group and the result was recorded as mean number of blood vessels per field.

8000 cells/well were seeded in 8-well chamber slides and, the following day, were treated for 24, 48, or 72 h with 50 nM calcitriol or solvent. Slides were fixated using 4% PFA before staining with the following primary antibodies: OLIG2 (R&D Systems, Minneapolis, MN, USA), GFAP (Dako Cytomation, Glostrup, Denmark) SOX2 (R&D Systems, Minneapolis, MN, USA) over night. Subsequently, slides were stained with a species-corresponding secondary antibody (goat anti-mouse (Alexa Fluor 594 F(ab’)2 fragment IgG (H+L)); (goat anti-rabbit (Alexa Fluor 488 F(ab’)2 fragment goat anti-rabbit IgG (H+L)); donkey anti-goat (Alexa Fluor 488 IgG (H+L))). Pictures were taken using a Nikon Eclipse TE2000-S fluorescent microscope operated by NIS elements software and adjusted using FIJI. Quantification was performed in Fiji by measuring the integrated density of the fluorescent signal (mean intensity of measured cell multiplied with the measured cell area) of 50 cells per condition using at least three pictures. The area was selected using the freehand selection tool, ensuring an exact and individual measurement for each cell. Violin plots were generated using GraphPad Prism 9.

Podocytes were plated on collagen coated 6 well tissue culture clusters and then differentiated as previously described^{64 (link)}. Expression of polymerized actin was visualized by phalloidin staining using Alexa Fluor 568 phalloidin from Molecular Probes (Eugene, OR), (product number A12380). For the experiments, podocytes were incubated in the indicated culture conditions for 24 hours. Cells were then fixed in 2% paraformaldehyde for 10 minutes and the excess aldehyde quenched with 0.1 M glycine in Dulbecco’s phosphate buffered saline (D-PBS). Cell were permeabilized with 1% Triton-X in D-PBS for 2 minutes and then incubated with Alexa Fluor 568 phalloidin (1:100) in Alexa Fluor 568 phalloidin for 30 minutes. After washing, cover slips were applied using adhesive containing DAPI (4′,6-diamidino-2-phenylindole) and slides examined using a Nikon Eclipse TE2000-S fluorescent microscope. Digital images were obtained with NIS Elements-F imaging software (version 3.2).