distance ± SEM measured in 6 independent experiments ( n = 6). The graph shows the average number on puncta represented as Log2 (SYTO foci + 1) vs. SYTO-positive discrete puncta were scored with the particle analyzer in 15 bins covering a distance of 150 μm from the edge of the soma. The longest Tau-positive neurite was selected with a segmented line and straighten, smoothen and binarized with the Ma圎ntropy mask (Ma圎ntropy). Images were converted to 8-bit and binarized with the Ma圎ntropy mask. (B) SYTO-positive staining from randomly selected cells was filtered with the convolver, brightness and contrast were adjusted. *** p < 0.001 * p < 0.05 n.s, not significant two-tailed t-tests. Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 ( n = 5, − SYTO negative samples compared to their corresponding + SYTO controls) or 6 ( n = 6, + SYTO + DNAse and + SYTO + RNAse compared to their corresponding + SYTO controls) independent experiments. To determine if SYTO selectively labeled RNA, some fixed cells were digested with DNAse (3, + SYTO + DNAse) or with RNAse (4, + SYTO + RNAse). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Puromycin-positive foci in axons are a result of local protein synthesis.
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