Fitc conjugated secondary antibody

An in situ analysis was used to detect miR-200c level in the human endometrium of proliferative and secretary phases, and mouse endometrium in non-pregnancy, pregnancy and pregnancy injected with miR-200c mimics groups. The tissues were fixed with 4% paraformaldehyde overnight and embedded in paraffin for sections (4 μm). The sections were deparaffinized following with proteinase K incubation (20 μg/ml) at 37 °C for 20 min. After washing, the sections were dehydrated and hybridized with biotin-labeled miR-200c probe (5′-TCCATCATTACCCGGCAGTATTA-3′, GenePharma) and the scrambled control probe (5′-TTGTACTACACAAAAGTACTG-3′, GenePharma) at 50 nM at 37 °C for 18 h. Then, the slides were incubated with FITC-conjugated secondary antibody (1:100, BOSTER, Wuhan, China) at 37 °C for 1 h. The signals of miR-200c were detected under a fluorescent microscope, and representative images were shown.

Expression levels of p65 and β-catenin were detected using immunofluorescence staining. Spheres were fixed in 4% paraformaldehyde at room temperature for 20 min, blocked in blocking buffer (PBS+2% BSA+0.3% Triton-100) at room temperature for 1 h, and incubated with primary antibodies against p65 and β-catenin (1:200, rabbit) at 4°C overnight. Cells were then treated at room temperature with FITC-conjugated secondary antibody (Boster Biological Technology) for 1 h and DAPI (Beyotime Institute of Biotechnology) for 30 min. Fluorescent signals were visualized using a Leica fluorescence microscope (magnification, ×200; Leica Microsystems GmbH).

Wound tissue samples from the BALB/c nude mice were collected on day 7 and 14 after injury, and fixed in 4% paraformaldehyde. All the tissues were embedded in paraffin blocks and sliced into paraffin sections. These sections were processed and stained with hematoxylin and eosin (H&E), and Masson staining. For immunofluorescence staining, anti-F4/80 (1:100, Servicebio, GB113373, China), anti-CD206 (1:100, Servicebio, GB113497, China), anti-CD31 (1:400, BOSTER, A01513-3, China) and anti-α-SMA (1:400, BOSTER, BM0002, China) antibodies were used. The following secondary antibodies applied were used: Cy3-conjugated secondary antibody (1:200, Servicebio, GB21303, China), FITC-conjugated secondary antibody (1:200, Servicebio, GB22303, China), Cy3-conjugated secondary antibodies (1:200, BOSTER, BA1032, China) and FITC-conjugated secondary antibody (1:200, BOSTER, BA1101, China). Wound tissue samples from the Bama miniature pig were collected on day 35, and fixed in 4% paraformaldehyde. All the tissues were embedded in paraffin blocks and sliced into paraffin sections. Then, the sections were then processed and stained with hematoxylin and eosin (H&E), and Masson staining. Images were captured with a microscope (Nikon, C2+, Japan) and a slide scanner microscope (Olympus, VS200, Japan), and analysed by the Image J software (NIH, Image J 1.8, USA).