Image pro plus software v7

To study cell adhesion, MC3T3-E1 cells were seeded (5 × 10⁴ cells/cm²) on the surface of samples from each group and incubated at 37°C in 5% CO₂. After 7 days of culture, cells on substrates were fixed and permeabilized using a Fir & Perm Reagent kit (Nordic MUbio, Susteren, The Netherlands). Then, blocking solution iBindTM Buffer (Invitrogen, Carlsbad, CA, USA) was applied for 45 min, and stained for 30 min with phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) that marks the cytoskeletal component of the cells. Cell nuclei were then counterstained with Hoechst 33342 (Thermo Fisher Scientific) for 5 min to highlight the nuclei of living cells. Cells were observed with an inverted fluorescence microscope (Olympus IX70, Tokyo, Japan), and images were analyzed with Image-Pro Plus software v7.0 (Media Cybernetics, Rockville, MD, USA).

The frozen kidney samples were cryosectioned and processed according to the methods of [6 (link)]. Briefly, kidneys were cryosectioned at five microns and mounted on glass slides. After fixation/permeabilization in −20 degree Celsius methanol, 0.3 µM 4’6-diamidino-2-phenylindole (DAPI) was applied to the sections in order to visualize all nuclei. An aqueous mounting medium-AquaPolymount (Warrington, PA, USA)-was then used to mount the sections under glass coverslips. Within 24 h, labeled sections were viewed using a 40× objective under phase contrast and fluorescent illumination on a Nikon Eclipse 400 fluorescent microscope. Total nuclei and ethidium-labeled nuclei were quantified within randomly-selected fields. There were approximately 200−300 cells in an area of 9.1 × 10⁴ µm² in each field. Viability analysis was performed on digital images from three fields per slide, four slides per animal. Digital images were captured with a 5.0 megapixel Evolution^TM MP digital camera by Media Cybernetics. Previously, we noticed that the staining intensity of ethidium homodimer and especially DAPI faded over time [6 (link)]. Therefore all the images of renal sections were captured within 48 h following DAPI staining. Digital images were processed using Image Pro Plus Software (v. 7.0) (Media Cybernetics, Silver Spring, MD, USA, 2009).