Joris van Dongen

134 Chapter 6 14. Wash samples with PBS three times and incubate samples with primary antibodies at room temperature for 60 min.: for α -SMA (1:200) + 1% BSA + 1% Human serum in PBS, for Perilipin A (1:200) + 1% BSA + 1% Human serum in PBS, for vWF (1:200) + 1%BSA + 1% Swine serum in PBS. 15. Wash samples with PBS three times. 16. Incubate samples with secondary antibodies for 30 min. at room temperature: polyclonal Rabbit anti-Mouse (1:100) + 1% BSA + 1% Human serum in PBS for α -SMA, polyclonal Goat anti-Rabbit (1:100) + 1% BSA + 1% Human serum in PBS for Perilipin A and polyclonal Swine anti-Rabbit (1:100) + 1% BSA + 1% Human serum in PBS for vWF. 17. Wash samples with PBS three times 18. Incubate α -SMA sample with a third antibody at room temperature for 30 min. with polyclonal Swine anti-Rabbit (1:100) + 1% BSA + 1% Human serum in PBS. 19. Wash α -SMA samples with PBS three times. 20. Incubate all samples with DAB for 10 min. at room temperature in the dark. 21. Wash all samples in demi water three times for 5 min. 22. Incubate all samples with hematoxylin for 1.5 min. 23. Rinse samples in water for 4 min. 24. Mount all the samples and place a coverslip (see Note 3). Masson’s Trichrome staining of tSVF 1. Follow step 1. till 9. of section 3.3. 2. Fix samples in 4% PFA for 60 min. 3. Permeabilize samples in 1% triton X-100 for 10 min. 4. Wash samples with PBS three times. 5. Fix samples in Bouins fixative at 51 °C. for 5 min. 6. Wash samples in demiwater until color disappears. 7. Incubate samples with Weighert’s iron hematoxylin for 20 min. 8. Wash samples in demiwater until color disappears. 9. Incubate samples with Beibrich Scarlet-acid fuchsine for 20 min. 10. Wash samples in demiwater for 2 min.