Joris van Dongen

133 Isolation of SVF by the FAT procedure 2. Wash the SVF with PBS three times. 3. Fix the SVF with 2% PFA for 30 min. 4. Wash the SVF with PBS three times. 5. Stain the nuclei DAPI in the dark for 30 min. 6. Wash the SVF with PBS three times. Histological characterization of SVF 1. Fix the isolated SVF in 10% formalin overnight at 4°C. 2. Embed the SVF in a pre-heated (60°C) 1.8% agarose solution (1:2). 3. Place the SVF/agarose solution at 4°C for 30 min. 4. Dehydrate samples with the following steps in sequence at room temperature: a. 50% alcohol for 30 min. b. 70% alcohol for 30 min. c. 96% alcohol for 30 min. d. Twice in 100% alcohol for 30 min. e. Twice in Xylol for 30 min. 5. Embed the samples in paraffin. 6. Cut four mm sections and deparaffinize them in xylol for 15 min. 7. Refresh the xylol and place the samples for another 10 min. in xylol. 8. Move the samples and place them in 100% alcohol for 10 min, then in 96% alcohol for 3 min. and finally in 70% alcohol for 3min at room temperature. 9. Wash the samples in demi water for 3 min. 10. Incubate samples overnight with 0.1MTris/HCl buffer (pH 9.0) at 80°C for α -SMA as well as for PerilipinA staining and with 10 mMTris/1 mMEDTAbuffer (pH 9.0) at 80°C for vWF. 11. Cool down to room temperature for 30 min. 12. Wash samples with PBS three times. 13. Endogenous peroxidase activity is blocked with 3% hydrogen peroxidase in PBS at room temperature for 30 min.