Mouse anti fibronectin

Representative sections were stained with hematoxylin and eosin (H & E) and Masson’s trichrome, and then examined by light microscopy. Keloid spheroid sections were incubated at 4 °C overnight with mouse anti-collagen type I (ab6308; Abcam, Ltd., Cambridge, UK), mouse anti-collagen type III (C7805; Sigma, St. Louis, MO), mouse anti-elastin (E4013; Sigma), mouse anti-fibronectin (sc-52331; Santa Cruz Biotechnology), rabbit anti-TGF-β1 (ab9758; Abcam, Cambridge, UK), mouse anti-EGFR (Ab-1; Oncogene Research Products, Calbiochem), rabbit anti-Erk 1/2 (#4370 S; Cell Signaling Technology, Beverly, MA), and rabbit anti-Smad 2/3 complex (#8685 S; Cell Signaling Technology) mouse anti-PCNA (DAKO), goat anti-Cytochrome c (SC-8385; Santa Cruz Biotechnology) primary antibody, and then incubated at room temperature for 20 min with the Dako Envision™ Kit (DAKO, Glostrup, Denmark) as secondary antibody. Diaminobenzidine/hydrogen peroxidase (DAKO, Carpinteria, CA) was used as the chromogen substrate. All slides were counterstained with Meyer’s hematoxylin. The expression levels of TGF-β1, EGFR, Erk 1/2, Smad 2/3, type I and III collagen, elastin, and fibronectin were semi-quantitatively analyzed using MetaMorph^® image analysis software (Universal Image Corp., Buckinghamshire, UK). Results are expressed as the mean optical density for six different digital images.

Before being used for cell culture, the micropatterned surfaces were coated with fibronectin to enhance cell adhesion. The micropatterned polystyrene plates were first sterilized by soaking in a 70% ethanol aqueous solution for 30 min, followed by rinsing with Milli-Q water 3 times. A total of 0.2 mL of 20 μg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA) in a NaHCO₃ (pH = 8.4) aqueous solution was spread on the micropatterned surfaces. After being incubated at 37 °C for 1 h, the micropatterned plates were washed with the NaHCO₃ aqueous solutions 3 times, and then further washed another 3 times with sterile Milli-Q water.
To characterize the fibronectin-coated micropatterned surfaces, immunological staining was performed using a mouse anti-fibronectin primary antibody (1:200 in 1% bovine serum albumin (BSA) in PBS, Santa Cruz Biotechnology, Dallas, TX, USA) and an Alexa Fluor-488 goat anti-mouse IgG (1: 1000 in PBS, Invitrogen, Waltham, MA, USA) as the secondary antibody. The stained micropatterned surfaces were observed under a fluorescence microscope.

Rat lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and then sliced into 5-μm-thick sections. Xylene was used for de-paraffinizing the paraffin-embedded sections, and a graded series of ethanol were used for processing the sections. Then, sections were stained with hematoxylin and eosin (H&E) sequentially. SA-β-gal staining was performed with Senescence β-Galactosidase Staining Kit (CST, Danvers, MA, USA) following the manufacturer’s instruction. The percentage of SA-β-gal positive cells was calculated and normalized to the control group. Immunofluorescence (IF) staining of rat lung tissue was performed as previously described [31 (link)]. The slides were stained with rabbit-anti-Bmi-1 (CST, 1:100 dilution), mouse-anti-α-SMA (CST, 1:100 dilution), mouse-anti-fibronectin (SANTA CRUZ, USA, 1:100 dilution), and DAPI (Sigma-Aldrich) and observed with fluorescence microscopy (OLYMPUS, Japan). Immunohistochemistry (IHC) staining of rat lung tissues was performed as previously described [32 (link)] and the slides were stained with fibronectin primary antibody (SANTA CRUZ, 1:50 dilution).