Buffered formalin

Methylene blue staining was used to visualize the attached L929 cells on the collagen-coated polystyrene culture plate and the PDMS 12 h after seeding. The cells were fixed with 10% buffered formalin (FUJIFILM Wako Pure Chemical Corporation) and stained with 1.4% methylene blue solution (Sigma-Aldrich) for 30 min at room temperature. After washing with 10 mM sodium borate buffer (Sigma-Aldrich), stained cells were observed under a light microscope.
The cell viability on collagen-coated polystyrene culture plates and PDMS 12 h after seeding was quantified using a tetrazolium salt-based assay (WST-1; Roche Diagnostics, Tokyo, Japan). For WST-1-based colorimetry, 10% (v/v) WST-1 reagent was added to the culture medium. The culture plate was incubated at 37 °C for 3 h, and then the supernatants were transferred into a 96-well microplate. The amount of formazan produced in the supernatant was measured using an ELISA plate reader at 450 nm.

A pair of Cauda epididymis from adult mice (11–17 weeks) were placed in 1 ml of D-MEM (Wako, 044-29765)/10% bovine serum albumin (BSA) in a 35 mm petri dish (Corning, 430165) and torn to pieces using two fine forceps for 3 min. After incubation at 32 ^oC for 20 min, 20 μl of sperm suspension was fixed in 480 μl 10% buffered formalin (Wako, 062-01661) at room temperature for counting.

Four human DA tissues were subjected to tissue staining and transcriptome analysis. Each DA tissue was divided into two pieces. One piece was fixed with 10% buffered formalin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for tissue staining. The other piece of tissue was prepared for microarray analysis as follows. After the adventitia was removed, each DA tissue was divided into two parts: the inner part and the outer part, which mainly contained IT and the tunica media, respectively, as indicated by the yellow dotted lines in Figure 1A. The tissues were immediately frozen in liquid nitrogen and stored at −80 °C until all patient samples were collected. Total RNA preparation and microarray analysis were performed as described previously [10 (link),11 (link)]. Briefly, the frozen tissues were disrupted by a multi-bead shocker instrument (Yasui Kikai, Osaka, Japan). After buffer RLT with β-mercaptoethanol was added to the tissues, they were sonicated to ensure the samples were uniformly homogeneous. Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Microarray experiments were carried out using a SurePrint G3 Human GE 8 × 60 K v2 Microarray (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol.

Cell appearances of hGFs and THP-1 cells on polystyrene and/or PDMS substrates were observed under a phase contrast microscope. Cell morphometries were performed on microscopic images of THP-1 cells using ImageJ software version 1.53t (National Institutes of Health, Bethesda, MD, USA) to evaluate aspect ratio and circularity.
The cytocompatibility of each PDMS substrate was evaluated via phase microscopy and water-soluble tetrazolium (WST)-1-based assay (Roche Diagnostics, Tokyo, Japan) for quantification of adherent cells in the hGF culture. For WST-1-based colorimetry, a 10% v/v WST-1 reagent was added to the culture medium. The culture plate was incubated at 37 °C for 3 h and then the supernatants were transferred into a 96-well microplate. The amount of formazan produced in the supernatant was measured using an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm.
Methylene blue staining was used to visualize the attached hGFs on the collagen-coated polystyrene culture plate and PDMS, 12 h after seeding. The cells were fixed with 10% buffered formalin (FUJIFILM Wako Pure Chemical Corporation) and stained with 1.4% methylene blue solution for 30 min at room temperature. After washing with 10 mM sodium borate buffer, stained cells were observed under a light microscope.