Explant culture was performed as described previously.60 (link) Briefly, first-trimester healthy human placental villi were dissected into 2–3 mm tissues, explanted in 24-well culture plates precoated with phenol red-free Matrigel substrate (Corning, NY, USA), and cultured in DMEM/F12 medium with 10% FBS. To investigate the effect of Prdx2 on the proliferation in placental villi, eleven placentas were used. Explants from each placenta were divided into three groups, one for control siRNA treatment, two for siPrdx2-1 and siPrdx2-2 treatment, respectively.
Whole mount immunofluorescent staining was performed as described previously.60 (link) Briefly, the explants cultured for 72 h together with matrigel were fixed by 4% paraformaldehyde. After blocking and permeabilization, the explants were incubated with primary antibodies against CK7 (Abcam and Cell Signaling Technology), Ki67 (Cell Signaling Technology) or Prdx2 (Abcam) at 4 °C for 24 h and fluorescent secondary antibody (Alexa Fluor 488-conjugated goat anti-Rabbit IgG, Alexa Fluor 594-conjugated goat anti-Mouse IgG) for 24 h. Finally, they were counterstained with Fluoroshield mounting medium with DAPI (Abcam) and then photographed using a fluorescence microscope (Leica DMi8 microscope, Leica Microsystems).
Whole mount immunofluorescent staining was performed as described previously.60 (link) Briefly, the explants cultured for 72 h together with matrigel were fixed by 4% paraformaldehyde. After blocking and permeabilization, the explants were incubated with primary antibodies against CK7 (Abcam and Cell Signaling Technology), Ki67 (Cell Signaling Technology) or Prdx2 (Abcam) at 4 °C for 24 h and fluorescent secondary antibody (Alexa Fluor 488-conjugated goat anti-Rabbit IgG, Alexa Fluor 594-conjugated goat anti-Mouse IgG) for 24 h. Finally, they were counterstained with Fluoroshield mounting medium with DAPI (Abcam) and then photographed using a fluorescence microscope (Leica DMi8 microscope, Leica Microsystems).
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