Picro sirius red solution

Example 11

Methods

The Sirius Red stain was used for examining the effect of the present Spiranthes sinensis extract in Collagen accumulation. Briefly, 3×10⁴ of cells per well were seeded in 12-well plates and are incubated in 5% CO2 at 37° C. overnight for confluence. Afterward, cells were treated with various concentrations (5, 20, and 50 μg/mL) of the present Spiranthes sinensis extract for 24 h, respectively. Cells were washed twice with PBS, and then fixed by 3.7% paraformaldehyde (pre-warmed) for 10 min. Picro-Sirius Red solution (ScyTek, Logan, Utah, USA) was stained at RT under the dark for 1 h. Cells were washed twice with ddH2O and counterstained with Hematoxylin (Sigma-Aldrich) for 1 min. Cells were washed with distilled water for three times and stored using glycerol. Images were acquired using ZEISS inverted microscope connected with Canon 700 D camera.

Data are expressed as means±S.E. Statistical comparisons of the results were made using one-way analysis of variance (ANOVA). Means within each column followed by the different letters were significantly different at p<0.05 by Turkey's test.

Results

The results are shown in FIG. 15. Both NHSCs and THSCs treated with Spiranthes sinensis extract (5, 20, and 50 μg/mL) for 24 h showed reduced perinuclear collagen comparing with the control groups.