Fluorescein conjugated secondary antibody

Cells were fixed by 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 5% bovine serum albumin (BSA) for 1 h. The samples were stained with primary antibodies against AIF (Santa Cruz Biotechnology, Santa Cruz, CA), p-Akt (pS473, Epitomics, Burlingame, CA), MnSOD, catalase, NQO1, Hmox1, Bad and Bax (abcam, Cambridge, MA) at a concentration of 1:100 overnight at 4°C, and then incubated with fluorescein-conjugated secondary antibodies (Santa Cruz Biotechnology) at 37°C for 30 min. The cells were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). All staining was examined under a confocal laser-scanning microscope.

Following treatment, cardiac fibroblasts were washed in PBS, fixed with 10% formaldehyde solution 10 min at room temperature, and permeabilized with 0.1% Triton X-100 PBS 10 min at room temperature. Then cells were blocked in 5% BSA solution at room temperature followed by 4°C overnight incubation with primary antibodies (Vimentin, a-SMA, Fibroblast specific protein 1, Troponin T, CD31 and MRTF-A: Abcam, Cambridge, MA, USA). After washing three times with 0.1% Tween-20 PBS, these cells were incubated with appropriate fluorescein–conjugated secondary antibodies (Santa Cruz Biotechnology) for 2 hours at room temperature, followed with nuclear staining by Hoechst 33258 pentahydrate 1 µg/ml (Invitrogen, Eugene, Oregon, USA). The fluorescence was examined and photographed using Leica fluorescence microscope.

Heart tissues obtained seven days post-MI were dehydrated in 30% sucrose PBS solution and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and snap frozen in drikold. Frozen tissue sections (6.0 µm thick) were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, blocked with 5% goat serum, followed by 4°C overnight incubation with primary antibodies (Collagen I: ab 292 Abcam, Cambridge, MA, USA) followed by incubation with respective secondary antibodies. After three time washing with 0.1% Tween-20 PBS, frozen slide were incubated with appropriate fluorescein–conjugated secondary antibodies (Santa Cruz Biotechnology) for 2 hours at room temperature, followed with nuclear staining by Hoechst 33258 pentahydrate 1 µg/ml (Invitrogen, Eugene, Oregon, USA). The fluorescence was examined and photographed using Leica fluorescence microscope.

Mouse eyeballs were excised and snap-frozen in Tissue-Tek OCT compound at 2 or 3 days after levobunolol, atenolol, or ICI 118, 551 treatment. The frozen sections (7 μm) were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with anti-CK3 (Abcam, Cambridge, MA), anti-CK14 (Abcam), anti-CK19 (Proteintech, Chicago, IL), anti-Ki67 (Abcam), anti- phosphorylated epithelial growth factor receptor (pEGFR, Abcam), or anti-phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2, Cell Signaling Technology, Danvers, MA) overnight at 4 °C and subsequently incubated with fluorescein-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, followed by 4′, 6-diamidino-2-Phenylindole (DAPI, Solarbio, Beijing, China) staining for 5 min. All stainings were observed through an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) and were quantified by Image J software. Firstly, the images were put on, then, which were conversed to 8 bit and adjusted to gray scale followed by measuring parameters including the area, mean gray value (Mean), integral optical density. Finally, we got average gray scale of these images which were used to statistically analyze.