Tissues were collected and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated, embedded in paraffin, and sectioned at 5 to 6 μm. Whole mount samples were fixed and dehydrated according to the standard protocol. Tissues were then cut into thin strips.
For in situ hybridization, tissues were hybridized with digoxigenin-labeled probes. Signals were detected using an anti-digoxigenin antibody coupled to alkaline phosphatase. Sfrp4 (nt 183-755), Follistatin (nt 409-1075), and Fgf7 (nt 486-1088) probes were obtained as follows: Wnt5a (nt 74-1156) (Dr. Sharpe, King's College London), Dkk1 (nt 112-942) (Dr. Millar, University of Pennsylvania), Lef-1 (1176-1905) (Dr. Thesleff, University of Helsinki).
Immunofluorescence staining was performed with anti-follistatin (mouse, 1:50, Abcam) and anti-perilipin A (rabbit, 1:100, Abcam) antibodies followed by secondary antibodies conjugated to Alexa-488 and Alexa-568.
For in situ hybridization, tissues were hybridized with digoxigenin-labeled probes. Signals were detected using an anti-digoxigenin antibody coupled to alkaline phosphatase. Sfrp4 (nt 183-755), Follistatin (nt 409-1075), and Fgf7 (nt 486-1088) probes were obtained as follows: Wnt5a (nt 74-1156) (Dr. Sharpe, King's College London), Dkk1 (nt 112-942) (Dr. Millar, University of Pennsylvania), Lef-1 (1176-1905) (Dr. Thesleff, University of Helsinki).
Immunofluorescence staining was performed with anti-follistatin (mouse, 1:50, Abcam) and anti-perilipin A (rabbit, 1:100, Abcam) antibodies followed by secondary antibodies conjugated to Alexa-488 and Alexa-568.