- For Frozen sections in OCT, the samples were cut into 5 μm thick slices, washed in distilled water for 10 min. For immunofluorescence, tissue sections were incubated in PBS at 5% milk for 60 min at room temperature. After a double washing in PBS for 10 min at room temperature, tissue sections were incubated overnight at 4°C with monoclonal anti-human antibodies (diluted 1:100 in PBS). Sections were washed in PBS three times for 10 min at room temperature and incubated for 90 min at 4°C with the secondary FITC- or PE-conjugated antibody (diluted 1:200 in PBS; AbCam, Cambridge, UK).
Tissue sections incubated for 90 min at 4°C only with conjugated secondary antibodies were used as negative controls. The sections were then observed under a fluorescence microscope (Nikon Instruments Italia, Calenzano, Firenze, Italy). The antibodies used were: anti-CD34 (Miltenyi-Biotech).
- Immunohistochemical analyses were performed with a DAKO CYTOMATION kit (En Vision+System-HRP-AEC; Dako Italia, Milan, Italy) according to the manufacturer's protocol. The antibodies used were anti-adiponectin (AbCam) and anti-PPAR-γ (AbCam).
Tissue sections incubated for 90 min at 4°C only with conjugated secondary antibodies were used as negative controls. The sections were then observed under a fluorescence microscope (Nikon Instruments Italia, Calenzano, Firenze, Italy). The antibodies used were: anti-CD34 (Miltenyi-Biotech).
- Immunohistochemical analyses were performed with a DAKO CYTOMATION kit (En Vision+System-HRP-AEC; Dako Italia, Milan, Italy) according to the manufacturer's protocol. The antibodies used were anti-adiponectin (AbCam) and anti-PPAR-γ (AbCam).