Immunohistological assay and Immunocytochemical assay

Placentas obtained from pregnant rats (12 dpc) were fixed with 4% paraformaldehyde, and 10 μm paraffin sections or Rcho-1 cells fixed with 1% paraformaldehyde were prepared. Immunostaining was performed with a rabbit anti-rat Gal-4 polyclonal antibody (Invitrogen. Carlsbad, CA) and a rabbit anti-rat LC3 polyclonal antibody (Wako, Tokyo, Japan), incubated at 4 °C overnight and developed with the Envision system (DAKO, Carpenteria, CA). Double immunofluorescence staining was performed in combination with a rabbit anti-rat Gal-4 polyclonal antibody (Invitrogen, Carlsbad, CA) and either a mouse anti-rat LC3 monoclonal antibody (MBL, Nagoya, Japan) or a mouse anti-rat Cdx2 monoclonal antibody (Abcam, Cambridge, MA), and then detected with anti-rabbit antibody Alexafluor 555 for Gal-4 and anti-mouse IgG antibody Alexafluor 488 for LC3 and Cdx2. Normal rabbit IgG was used as a negative control. Observation was performed using a confocal microscopy. Immunocytochemical assay was performed using Rcho-1 cells on day 1 after induction of differentiation. And then, cells were fixed and incubated with fluorescence antibodies as described above. The stained cells were observed using a confocal microscopy.