Joris van Dongen

69 FAT procedure to obtain SVF for regenerative purposes nuclei. As a dead cell control a few samples were fixed with 2% PFA in PBS and then stainedwith PI. Resultswere evaluatedwith a confocal immunofluorescencemicroscope (Leica TCS SP2 AOBS spectral confocal microscope). Figure 1. The mechanical dissociation procedure results in four different fractions: (1) oily fraction, (2) SVF and (3) infiltration fluid fraction containing (4) a small pellet fraction. Immunohistochemistry & Masson’s Trichrome Four of the obtained samples of FAT-SVF and control were formalin-fixed and embedded in paraffin. 4 µm slides were deparaffinized and incubated overnight with 0.1 M Tris/HCL buffer (pH 9.0) for α -Smooth Muscle Actin ( α -SMA) and Perilipin A, von Willebrand Factor (vWF) staining was pre-incubated with 10 mmol Tris/1 mmol EDTA buffer (pH 9.0). Then, endogenous peroxidase activity was blocked with 30% hydrogen peroxide in PBS for 30 min RT. Samples were washed with PBS three times and incubated with primary antibody and 1% BSA in PBS for 1 hour RT. 1% human serum (HS) for α -SMA and Perilipin A and 1% swine serum for vonWillebrandt (vWF) was added to the primary antibody and 1% BSA. Negative controls were incubated without primary antibodies. Subsequently all samples, including negative controls, were washed with PBS three times and incubated with secondary antibody, 1% BSA, 1%HS in PBS for 30 min RT. Only a third antibody in 1% BSA and 1%HS in PBS for 30 min was used in α -SMA staining. Staining was completed with 3,3’-Diaminobenzidine (DAB) incubation (Sigma® Life Science, St. Louis, MO, USA) and hematoxylin