Inverted confocal dmi6000cs

NSCs were grown on sterile glass coverslips, treated as described for each experiment, followed by washing with PBS and fixing with 4% paraformaldehyde in PBS for 20 min at room temperature. After blocking with PBS containing 1% BSA, permeabilizing with 0.1% Triton X-100 and Glycine 1 M for 30 min, cells were washed with PBS and stained by indirect immunofluorescence using the antibodies described before. Samples were mounted with Prolong Gold Antifade (P-36930, Life Technologies) and randomly chosen field images were obtained in an Inverted Confocal DMI6000CS (Leica, Wetzlar, Germany) fluorescence microscope.

Cells were fixed with 4% paraformaldehyde and mitochondrial pattern was observed by TOM20 staining. Images were acquired with Inverted Confocal DMI6000CS (Leica, Wetzlar, Germany) fluorescence microscope. For analysis, we used the Mitochondrial Network Analysis [3 (link)] toolset, a combination of different ImageJ macros that allows the semi-automated analysis of mitochondrial networks in cultured mammalian cells [30 (link)]. Briefly, the image was converted to binary by thresholding following the conversion to a skeleton that represents the features in the original image using a wireframe of lines one pixel wide. All pixels within a skeleton were then grouped into three categories: end point pixels, slab pixels and junction pixels. The plugin analyzes how the pixels are spatially related and defined to measure the length of each branch and the number of branches in each skeletonized feature as well as the mitochondrial network morphology. The parameters used in the study were 1) Individuals, punctate, rods and large/round mitochondrial structures; 2) Networks, mitochondrial structures with at least a single node and three branches; 3) the mean number of Branches per Network; and 4) the average of length of rods/branches. 100 cells per each independent experiment were used to quantify the pattern of mitochondria.