Immunohistochemistry. For immunohistochemistry, embryonic (E) and postnatal (P) pituitaries from Wistar–Imamichi rats were fixed with 4% paraformaldehyde (PFA) in 50 mM phosphate-buffered saline (PBS), pH 7.5, maintained overnight at 4 °C, followed by substitution with 30% sucrose in PBS. Cryosections of 10 μm thickness were reacted with primary
BI2536 at the appropriate dilution at room temperature overnight. Primary antibodies used were guinea pig antiserum against rat PROP1 (1:1000, in the present study as described in the Supplemental materials and methods), rabbit antiserum against rat PIT1 (1:250, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat antiserum against human SOX2 (1:500 dilution, Neuromics, Edina, MN, USA), rabbit antiserum against pituitary hormones (ovine LHβ (1:2000 dilution), rat TSHβ (1:2000 dilution), rat GH (1:8000 dilution) and rat PRL (1:1500 dilution), which were kindly provided by the National Institute of Diabetes and Digestive and Kidney (NIDDK) through the courtesy of Dr. A.F. Parlow, and human ACTH (1:1000 provided by Dr. S. Tanaka at Shizuoka University, Shizuoka, Japan). After washing with PBS, incubation with secondary
antibodies was then carried out using FITC- or Cy3-conjugated AffiniPure donkey anti-guinea pig, rabbit and goat IgG (Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor 488 conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR, USA). The sections were washed with PBS and then enclosed in VECTASHIELD mounting medium with DAPI (Vector, Burlingame, CA, USA). Immunofluorescence was observed under fluorescence microscopy with a Confocal Laser Scan microscope; CLSm (Carl Zeiss, Oberkochen, Germany) and BZ-8000 (KEYENCE, Osaka, Japan).
