Donkey secondary antibodies

For immunohistochemistry, perfused aortas were post-fixed in 4% PFA overnight, then cryosectioned at 8 μm and stained with primary antibodies recognizing PECAM (BD Bioscience 553370, 1:100), αSMA (Sigma F3777, 1:250), MOMA-2 (AbD Serotec MCA519G, 1:100), F4/80 (Abcam ab6640, 1:100), SMMHC (Biomedical Technologies Inc. BT-562, 1:100) or Sm22α (Abcam ab10135, 1:100), with appropriate donkey secondary antibodies (Jackson ImmunoResearch). Immunofluorescence microscopy was performed with a Nikon Eclipse 80i microscope connected to a digital camera. Final image processing was performed with Photoshop (Adobe). For histological stains, perfused aortas were post-fixed with Bouin’s solution, paraffin embedded, and sectioned at 5 μm before staining with Movat’s pentachrome, Masson’s trichrome, or Prussian blue. Formalin-fixed aortic roots were cryosectioned at 10 μm and stained with Oil red O.

Brains were fixed by transcardiac perfusion followed by 1 hr of postfixation on ice with 4% formaldehyde/PBS solution. Brains were rinsed with PBS and cryoprotected by using 30% sucrose/PBS solution overnight at 4°C. Cryosections were prepared at 20 μm (for counting immunoprofiles) thickness. Immunohistochemistry was performed by using a PBS solution containing 1.5% normal goat serum and 0.25% Triton X-100 for all procedures. The washing steps were done with PBS. The sections were incubated overnight (ON) at 4°C with selected antibodies, followed by incubation at 4°C ON with donkey secondary antibodies (Jackson ImmunoResearch Laboratories).
For cell counting and post hoc examination of marker expression, sections were stained using rat anti-GFP, mouse anti-Reelin (1:500, MBL, D223-3), rabbit anti-dsRed (Abcam, ab62341), rabbit anti-VIP (1:1,000, Immunostar, 20077), Gt-PV (1:500, Swant:PVG214), and rat anti-SST (1:500, Millipore). For the analysis of nuclear localization of NFATc4 protein, rabbit NFATc4 (Abcam, ab62613, 1:500) was used.