LacZ staining

X-Gal Stock solution: 40mg/mL X-Gal in dimethylsulfoxide. 10mL were prepared and stored at 1mL aliquots at −20°C.

X-Gal staining solution: 2mM MgCl₂, 2.5mM K₃Fn(CN)₆, 2.5mM K₄Fn(CN)₆ in PBS pH 7.4. 500mL were prepared and stored in the dark at RT. Before use, X-Gal stock solution (40mg/mL) was added to a final concentration of 1mg/mL of X-Gal. Depending on the amount of tissue to be stained, 2 to 20mL of X-Gal staining solution per mouse were prepared.

X-Gal staining of whole-mount aortae or paraffin embedded sections of aortae was carried out as described (Feil et al., 2014b; Thunemann et al., 2017): Animals were transcardially perfused with Heparin-PBS (PBS with 250 mg/L heparin) followed by PBS containing 2% formaldehyde and 0.2% glutaraldehyde. Explanted aortae were fixed for 20 minutes in the same fixative solution at room temperature, washed twice with PBS for 5 minutes under gentle shaking and incubated overnight at room temperature in the dark under gentle shaking in X-Gal staining solution. After staining, aortae were again washed three times with PBS for 5 minutes under gentle shaking and stored at 4°C in 70% ethanol. Images for analysis were taken using a digital camera attached to a stereo microscope (Zeiss, Oberkochen, Germany). For paraffin embedded sections, aortae were dehydrated in increasing concentrations of ethanol (70%, 80% and 95%) 45 min each, followed by three times absolute ethanol, 1 h each. Tissue was cleared twice in Roti-Histol (for 30 min and for another 15 min) Aortae were immersed in paraffin 3 times for 1 h to overnight and embedded in paraffin. The aorta was placed in a small mold in appropriate orientation for cross-sectioning and the paraffin block was sectioned at 6 μm thickness using a microtome. Then, the aortae were deparaffinised as follows: Roti-Histol (2 x for 3 min), 100% ethanol (2 x for 3 min), 95% ethanol (1 x for 3 min), 70% ethanol (1 x for 3 min). Then, slides were rinsed with cold tap water. X-Gal-stained paraffin sections were co-stained using a standard H and E staining protocol.

LacZ (blue) area and total surface area of explanted aortae were measured using ImageJ and calculated as a percentage of the ratio of LacZ/total surface area.