Vector red ap substrate kit

TMA sections were deparaffinised, rehydrated and washed in distilled H₂O. After antigen retrieval in decloaking chamber (Biocare Medical) at 110 °C for 5 min in pH 10.0 retrieval buffer, (for PDGFR-β) or pH 9.0 (for α-SMA), sections were incubated with blocking solution for 30 min and then incubated overnight with antibodies against PDGFR-β (No. 3169 Cell Signaling Technology, Danvers, MA, USA at dilution 1 : 100) or α-SMA (Clone 1A4; Dako, Inc., Denmark at dilution 1 : 300). Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Rabbit IgG MP-5401 or ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and developed with Vector Blue AP Substrate Kit (SK-5300, Vector Laboratories).
To destroy alkaline phosphatases activity, sections were subsequently heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 buffer, and then incubated with blocking solution for 30 min. Primary antibody against CD34 (Clone JC70A) at a 1 : 100 dilution was added for overnight incubation at 4 °C. Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories) for 1 h at room temperature and developed with Vector Red AP Substrate Kit (SK-5100, Vector Laboratories).

Each heart was cut along a horizontal plane between the lower tips of the right and left atria. The upper portion was snap-frozen in OCT compound (TissueTeck). Sections (5 µm intervals) were stained with oil red O to detect lipid deposition. Other sections were incubated overnight with antivascular cell adhesion molecule-1 (VCAM-1) antibody, anti-intercellular adhesion molecule-1 (ICAM-1) antibody (Abcam), or antimacrophage antigen-3 (Mac-3) antibody (BD Biosciences). Thereafter, sections were incubated with biotinylated secondary antibody (VECTOR Laboratories, Inc.), followed by VECTASTAIN ABC-AP Kit (VECTOR Laboratories, Inc.) and were stained using a VectorRed AP Substrate Kit (VECTOR Laboratories, Inc.). All sections were counterstained with hematoxylin. The ratio of positive area to plaque area was then calculated^{28 (link))}.

RNAi-mediated knockdown was performed with 40 nM siRNA pools using Lipofectamine RNAiMAX (Invitrogen) in secondary 1B MEF cells or mESCs upon seeding as previously described (Samavarchi-Tehrani et al. 2010 (link); Golipour et al. 2012 (link)). After 24 h, Dox was added and replaced daily. Five days later, cells were fixed and stained for AP activity (Vector red; AP substrate kit, Vector Laboratories) and counterstained for DAPI. All siRNAs are listed in Supplemental Table S7.