Sc 122

Brain tissues were isolated from 8 weeks old Fgfr2^lox/lox (control), heterozyote Nestin-Cre;Fgfr2^+/lox and homozygote Nestin-Cre;Fgfr2^lox/lox mice, and homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Sodium deoxycholate, 1 mM EDTA, and Complete protease inhibitors (Roche)). Total protein concentration was determined with the Pierce BCA Protein Assay (Thermo Fisher Scientific), and 50 µg total protein per sample were separated in 10% NuPAGE Novex precast gels (Invitrogen) and blotted onto PVDF membranes (Hall/USA). Blots were blocked in 4% skim milk in TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) and probed with rabbit anti-Fgfr2 (1∶300; sc-122) and anti-hypoxanthine guanine phosphoribosyl transferase (Hprt) (1∶400; FL-218) antibodies (both from Santa Cruz Biotechnology). Membranes were developed in ECL substrate and exposed to Hyperfilm ECL (GE Healthcare).

Formalin-fixed and paraffin-embedded human placental tissue sections were de-paraffinized in xylene and rehydrated in descending concentrations of ethanol. The tissue sections were then subjected to antigen retrieval by heating for 10 minutes at 120°C in citrate buffer (pH 6) and allowed to cool down to room temperature. The tissue sections were treated with 0.3% H₂O₂ to block endogenous peroxidases and with goat or rabbit serum to block nonspecific antibody binding sites on PLAC1 and FGFR2IIIb or FGF7, respectively. Samples were incubated with primary antibodies against PLAC1 (mouse monoclonal, mu37, Ganymed Pharmaceuticals AG), FGFR2 (rabbit polyclonal, sc-122, Santa Cruz Biotechnology), or FGF7 (polyclonal goat, AF-251-NA, R&D Systems) overnight at 4°C; the sections were washed three times with phosphate-buffered saline (PBS) and then incubated with horseradish peroxidase-conjugated secondary antibodies, goat-anti-mouse, goat-anti-rabbit, or rabbit-anti-goat (Immunologic, bv, Duiven, Netherlands), respectively. For visualization of PLAC1, FGFR2IIIb, and FGF7 localization, the Vector NovaRED peroxidase substrate kit (Vector Laboratories Inc. Peterborough, UK) was used and nuclei were counterstained with hematoxylin/eosin. Tissue sections were dehydrated and mounted (X-TRA-Kitt, Medite, Burgdorf, Germany) prior to microscopic evaluation.