Journal: PLoS ONE

Article Title: Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity

doi: 10.1371/journal.pone.0148064

Figure Lengend Snippet: On day 8, washed PLTs from DT- or saline-treated mice were resuspended in Tyrode’s albumin/DMEM medium and incubated at 37°C for different periods of time. (A) PLTs were incubated for 0, 2, 4, 6 and 24 h. The percentage of TO bright PLTs, determined at 0, 6 and 24 h, was 82, 33 and 2%, respectively. At each time points, PLT samples were fixed and permeabilized, stained with the anti-rRNA mAb Y10b and A647-conjugated anti-mouse IgGs, counterstained with an A488-conjugated anti-GP1bβ mAb and analyzed by confocal microscopy. PLT outlines, determined by Photoshop analysis of anti-GP1bβ staining, are depicted. Representative micrographs of analyzed cells from DT-treated animals (+DT) at times 0, 4 and 24 h and from saline-treated animals (-DT) at time 0 are shown. (B) The mean fluorescence intensity per unit area in each PLT stained with Y10b (+) or an isotype-matched control antibody (C) (y-axis, log2 scale) was calculated as described in the methods; 500 to 550 PLTs from DT-treated mice (+) or saline-treated mice (-) were analyzed for each condition. A one way ANOVA analysis followed by a Bonferroni post-hoc test comparing each pair of conditions gave p values of <0.001 (continuous bars) or <0.01 (dashed bars). Other combinations of Y10b stained samples were not statistically different.

Article Snippet: The anti-rRNA mAb Y10b (5 μg/mL, mouse IgG3, Novus Biologicals) was used to label ribosomes and revealed with A647-conjugated goat anti-mouse IgG.

Techniques: Saline, Incubation, Staining, Confocal Microscopy, Fluorescence, Control