Anti shh igg1

Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min followed by a 2X wash with PBS and later permeabilized using 0.25% Triton X‑100. Cells were then blocked in 10% BSA for 1h followed by overnight incubation with rabbit monoclonal anti-Shh (IgG1, 1:500, Santa Cruz Biotechnology) or anti-Gli1 (IgG, 1:500, Abcam) antibodies at 4°C. After three washes with PBS, the mixtures were then incubated in the dark for 1 h with Alexa Fluor 488-conjugated donkey anti-rabbit, IgG secondary antibodies (1:200, Invitrogen). After three additional washes, nuclei were stained with 4’, 6-diamidino-2-phenylindole (Invitrogen) for 5 min. Immunofluorescence images were obtained by a Leica TCS SP8 laser scanning confocal microscope (Leica, Mannheim, Germany).

Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, then washed twice with PBS, and permeabilized with 0.25% Triton X-100. After incubation with 5% BSA for 1 h to block the nonspecific antibody binding, cells were stained with rabbit monoclonal anti-Shh (IgG1; 1 : 500; Santa Cruz Biotechnology) or anti-Gli1 (IgG; 1 : 500; Abcam) antibodies at 4°C for 10 h. Following three times washing in PBS for 5 min, the cells were incubated at 37°C for 1 h with Alexa Fluor 488-conjugated donkey anti-rabbit, IgG (1 : 200; Invitrogen Life Technologies) secondary antibodies. The preparations were then washed with PBS three times for 5 min each in the dark. Nuclei were stained with DAPI (Invitrogen) for 5 min. Immunofluorescence images were obtained using a Leica TCS SP8 laser scanning confocal microscope (Leica, Mannheim, Germany).