Spinning disk confocal imaging of MG-63 cells cultured onto (a) uncoated, (b) Ta-coated, (c) TaC-coated, (d) and TaC/a-C-coated samples at 48 h.

<p>The blue fluorescent-stained nucleus was stained using Hoechst 33258 dye. Cell density, 2,000 cells/cm²; original magnification, ×200.</p

Surface SEM micrographs of the Ta, TaC, and TaC/a-C coatings.

<p>Surface SEM micrographs of the Ta, TaC, and TaC/a-C coatings.</p

Morphology and distribution of MG-63 cells cultured onto uncoated, Ta-coated, TaC-coated, and TaC/a-C-coated samples for 72 h by using an inverted optical microscope.

<p>Cell density, 5000/cm²; original magnification, ×40.</p

MTT assay test of MG-63 cells incubation with the uncoated, Ta-coated, TaC-coated, and TaC/a-C-coated samples at day 5.

<p>Data show the mean and SD values. Each specimen was n = 6, and the assay was performed duplicates. *Significantly different from the control group (<i>p</i><.01).</p

XRD spectra of the deposited Ta, TaC and TaC/a-C coatings.

<p>XRD spectra of the deposited Ta, TaC and TaC/a-C coatings.</p

Figure 1

<p>(a) Ta4f and (b) C1s XPS spectra of the deposited TaC and TaC/a-C coatings.</p

RVC does not affect tissue morphology or the expression and distribution of proteins in tissue samples.

<p>(A) Morphological features of murine liver tissue samples stored in different preservative solutions. (B) The abovementioned paraffin-embedded tissue samples were sliced and stained with H&E for the examination of cellular morphological characteristics. RNAlater and 100% ethanol caused significant tissue dehydration, whereas the RVC-based preservative solution did not cause this phenomenon. (C-D) Determination of EGFR and PCNA protein expression and distribution by immunohistochemical staining in tissue samples treated with different preservative solutions. The results from the RVC-based groups were consistent with the data of the control group.</p

The RVC-based preservative solution has a milder effect on protein activity than 100% ethanol and RNAlater.

<p>(A-B) Determination of the effect of different preservative solutions on intracellular luciferase activity. The lysate of cells expressing luciferase was reacted with different preservative solutions, and luciferase activity was measured after 0, 0.5, 1, and 3 hours (n = 3). *** p < 0.001 compared to the NT group. (C) The effects of various preservative solutions on intracellular GFP activity. The RVC-based preservative solution exerted a significantly milder effect on GFP fluorescent activity than 100% ethanol and RNAlater.</p

Different types of preservative solutions cause changes in the microenvironment of cells and affect gene expression levels.

<p>(A, B) RNA was isolated from tissue samples immersed in different preservative solutions at −80°C for 1 month and subjected to whole-transcriptome analysis. Different preservative solutions affected the gene expression levels within tissue samples to different degrees. The total number of genes affected by the different preservation solutions compared to the control group are shown in (C). (D) The RNA from the abovementioned samples was analyzed for the expression of 12 miRNAs by real-time RT-PCR using <i>snoRNA202</i> as an internal control. Different preservative solutions also affected the expression of miRNA in the tissue samples. * p < 0.05, ** p < 0.01, *** p < 0.001 (compared to the NT group). (E) The genomic DNA was isolated from the samples processed as described above and subjected to PCR for amplification of an <i>IFN-r</i> gene fragment. (F) The PCR-amplified <i>IFN-r</i> gene fragments were subjected to sequence analysis by the Sanger sequencing method. The correct sequences were obtained from the samples stored using all preservation methods.</p

Both the RVC-based preservative solution and RNAlater can effectively delay RNA degradation.

<p>(A-B) Quality of RNA in tissue samples (n = 3) preserved with different concentrations of RVC in the preservative solution at 4°C or −80°C for 7 days. (C) Quality of RNA in tissue samples (n = 5) preserved in different preservative solutions at 4°C for 7 days and −80°C for one month (D). NT: stored without any preservative solution; RVC+G: RVC+15% glycerol; RVC+D: RVC+5% DMSO; * p < 0.05, ** p < 0.01, *** p < 0.001 (compared to the NT group).</p