Scion image for windows

Immunoblotting was performed to measure the expression of proteins, as previously described^{43 (link)}. VSMCs were stimulated with PDGF-BB (25 ng/mL) for 5 min for ERK1/2 and PLCγ1, 10 min for JNK and p38, and 15 min for STAT 3 and Akt phosphorylation assays. To measure the expression of CDK2, CDK4, cyclin D1, cyclin E₁, p21, p27, and proliferating cell nuclear antigen PCNA, and the phosphorylation of Rb, VSMCs were stimulated with PDGF-BB (25 ng/mL) for 24 h. The levels of each protein were normalized to the levels of β-actin or the respective total protein. Band intensities were quantified using Scion Image for Windows (Scion Corp., Frederick, MD, USA).

Autoradiograms from western blotting experiments were digitized using a Dia-Scanner (Epson Perfection 4870 PHOTO). Optical density values were measured using NIH Scion Image for Windows (alfa 4.0.3.2; © 2000–2001 Scion Corporation). Biochemical data were analyzed using one-way ANOVAs followed by Newman–Keuls post hoc test. Data from Amperometry were acquired and analyzed with the pClamp 9 or pClamp 10 software (Axon Instruments). Data are expressed as % of the baseline response measured for each slice during the 5–10 min preceding start of perfusion with T₁AM. Statistical significance of the results was assessed by using Student’s t-test for paired observations (comparisons with baseline within single groups) or one-way ANOVA multiple comparison test followed by Newman–Keuls post hoc test since the samples from WT and KO mice were loaded in separated gels. The numbers of individual replicates are shown in the graphs, while p-values, degrees of freedom and F values are detailed in the results part.

Cerebellar white matter demyelination was examined with the KB method without Nissl staining^{33 (link),35 (link)}. Eight sagittal sections of the cerebellar hemispheres were prepared at 200 µm intervals at the thickness 5 µm, stained with KB. These eight sections were tile scanned of the entire cerebellar hemisphere at 400 × magnification in each mouse using 9 animals per time course. The Luxol fast blue intensity, which stains myelin (fast blue) in the white matter, was then semi-quantified to evaluate demyelination. These areas were manually traced with blinded investigators and converted to black and white; black areas were semi-quantified using Scion Image for Windows (Scion Corporation, USA). The pixels of the black myelinated regions were divided by the pixels from the total traced areas and expressed as percentages. Eight serials of sagittal sections were averaged for each animal. These procedures were performed by two investigators (H.Y. and K.Y.) who were blinded to the experimental groups.

For quantification of presumptive interactions among neurons, astrocytes, and microglia, four sections (43672.47 μm²) from five animals were selected from each group. For each section, three microscopic fields extending through the rostrocaudal dimension of the nucleus were sampled using a 40X objective. The colocalization area (NeuN/Iba1 or NeuN/GFAP) was estimated from representative z- stack confocal microscopy images by using the public domain image analysis software Scion Image for Windows (version beta 4.0.2; developed by Scion Corp), as previously described (Fuentes-Santamaria et al., 2008 (link)). The immunostained profiles above the threshold were identified and the resultant images were converted into binary. To determine the degree of colocalization, the binary images corresponding to a given antibody were pseudocolored (green for Iba1 or red for GFAP) and then merged with the other pseudocolored image (blue for NeuN). The resultant image was used to quantified the overlapped area (putative region of interaction). The percentage of variation was calculated as above mentioned:
Where AEC and AUC represent the immunostained colocalized area in the noise-exposed and unexposed conditions, respectively.

Cells were counted in segments of ~250 μm in length each along the basilar membrane by using the public domain image analysis software Scion Image for Windows (version beta 4.0.2; developed by Scion Corp). Dark spots and/or the phalangeal scars of supporting cells in the spaces previously occupied by OHC were used as criteria to assess sensory cell loss (Hu and Henderson, 1997 (link); Minami et al., 2007 (link); Fetoni et al., 2013 (link)). The results were expressed as percentage of remaining OHC compared to control condition, over the length of the basilar membrane (Viberg and Canlon, 2004 (link); Fetoni et al., 2013 (link)). Image acquisition was performed with a laser scanning confocal microscope (LSM 710; Zeiss, Germany). By using the ZEN 2009 Light Edition software (Zeiss, Germany), the images of each dye were captured sequentially with a 40X Plan Apo oil-immersion objective (1.4 NA), merged and saved as TIFF files.