Goat anti rabbit alexa 555

DMEM medium and cell culture supplements were purchased from Sigma (Steinheim, Germany). Cell culture plastics, phosphate-buffered saline (PBS), and fetal calf serum (FCS) were purchased from PAA Laboratories GmbH (Pasching, Austria). DNase I (RNase-free) and First Strand cDNA Synthesis Kit were purchased from Fermentas (Ontario, Canada). 5-AZA and L-ascorbic acid 2-phosphate were obtained from Sigma-Aldrich (Steinheim, Germany). All other chemical compounds were purchased from Carl Roth (Karlsruhe, Germany). 5-hmC (39769) mouse mAB was purchased from Active Motif (Carlsbad, CA, USA). Proliferating cell nuclear antigen (PCNA) (ab92552) rabbit mAB was obtained from Abcam (Cambridge, UK). Corresponding secondary antibodies goat anti-rabbit Alexa 555 and goat anti-mouse 488 were acquired from Invitrogen (Carlsbad, CA, USA). Anti TET2 (SAB3500711), anti TET3 (SAB2700682), and anti glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G9545) antibodies were used from Sigma (Munich, Germany). GADD45B (SC33172) rabbit mAB was from Santa Cruz, and Snail (3879) rabbit mAB, E-cadherin (14472) mouse mAB, p21 (2947) rabbit mAB, cyclin-B1 (4138) rabbit mAB, and HRP-linked anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell Signaling (Beverly, MA, USA).

The cells were placed on coverslips and exposed to TGF-β1 (5 ng/mL) after transfection with miR-29b mimic, miR-29b inhibitor, or siHSP47. Subsequently, The cells were permeated with 0.2% Triton X-100 in 1% bovine serum albumin for 10 min, blocked by 5% bovine serum albumin for 1 h at room temperature, incubated overnight at 4 °C with monoclonal anti-HSP47, anti-E-cadherin, and anti-vimentin antibodies (Sigma-Aldrich), then fixed with 4% paraformaldehyde. Goat anti-mouse Alexa 488 (Invitrogen) and goat anti-rabbit Alexa 555 (Invitrogen) secondary antibodies were added to the cells for incubation. Finally, the nucleus was counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen) and the stained cells were visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).

Each stage of embryos without zona pellucida was fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed samples were permeabilized using 1% Triton X-100 for 1 hour at room temperature and washed three times with phosphate-buffered saline (PBS). The embryos were blocked using 10% goat serum or donkey serum in PBS for 1 hour at room temperature. Samples were stained with anti-GFP (5 μg/mL), RFP (1 μg/mL), and anti-OCT4 (1 μg/mL) or anti-SOX2 (2.5 μg/mL) in PBS containing 10% goat serum at 4°C overnight (Table S1). After washing 3 times in washing solution (PBS with 0.2% Tween-20 and 1% BSA for 10 min), embryos were incubated with goat anti-mouse Alexa 647 (Invitrogen, Carlsbad, California, USA), goat anti-chicken Alexa488 (Invitrogen), and goat anti-rabbit Alexa555 (Invitrogen) in PBS with 10% goat serum or donkey serum at RT for 1 hour. All samples were washed 3 times with washing solution after secondary antibody treatment. Immunostained embryos were mounted on a glass slide with Prolong Gold and DAPI (Invitrogen) and cured for more than 24 hours. We described the list of antibodies in Table S1. Images of stained cells were captured using a confocal microscope and processed by the ImageJ program.