Eclipse ti u instrument

Extirpated penises were fixed in 10% paraformaldehyde, which was replaced with 10%, 20%, and 30% sucrose solutions. The penis samples were then frozen in O.C.T. compound (Sakura Finetek Japan Co. Ltd). Seven‐micron cross‐sections were cut and used for Masson's trichrome staining. For quantitative image analysis, stained sections were photographed using an Eclipse Ti‐U instrument (Nikon Corp.) and NIS‐Elements D30 (Nikon). Saved images were analyzed using Photoshop CS4 (Adobe Systems Inc.). Red and blue pixel numbers in half of the CC tissue samples were analyzed for smooth muscle (SM) (red) and collagen (blue) staining, and the SM to collagen ratio was calculated.

Mice mBMSCs were cultured with medium (High-Dulbecco’s modified Eagle Medium containing 10% fetal bovine serum). Cells in passages 3–5 were used for the following assays. The Ti samples (10 mm × 10 mm) were placed in 24-well plate, to which mBMSCs (3 × 10⁴ cells per well) were added. In the fluorescence-based assay, after 24 h, the Ti samples were cleaned with PBS, and treated with 4 vol% neutral paraformaldehyde solution at 4 °C overnight. Then, the Ti samples were treated with 0.1% Triton for 12 min, F-actin solution (10 mg/L) for 60 min, and DAPI solution (5 mg/L) for 6 min. After that, the samples were cleaned with PBS and characterized with the Eclipse Ti-U instrument (Nikon, Japan) under the FITC and DAPI channels. To characterize the distribution of the cells on the gradient surface, we obtained 9 images along the gradient direction of the sample, then combined them into one image, and divided the combined image into 10 bands.
The biocompatibilities of the Ti samples were characterized by the CCK-8 assay. After being cultured with the cells for 24 and 72 h, the Ti samples were transferred to new 24-well plates. Then, the complete medium (350 μL) and the CCK-8 solution (35 μL) were added for each Ti sample. After 3 h of culturing, the OD value of the solution (100 μL) was characterized at the wavelength of 450 nm.