Oct compound

We detected the content of nitrotyrosine in frozen sections by immunofluorescence technique, and the mouse heart tissue was embedded in OCT compound (Bio-Optica, Milan, Italy). The 7 um tissue sections were prepared and incubated with rabbit anti-nitrotyrosine (R&D, USA) for 2 h at room temperature. After washing with PBS, the fluorescence in-conjugated secondary antibody was added for 1 h. finally the nuclei were counterstained with DAPI. The fluorescently stained slides were observed under an Olympus fluorescence microscope (Tokyo, Japan), and fluorescent images were taken.

Capillary density and inflammatory cells were quantified within gastrocnemius muscles using immunofluorescence analysis. Muscle samples were embedded in OCT compound (Bio-Optica) and processed as previously described^{66 (link)}. Cryosections of the ischemic limbs were stained with rat anti-mouse CD14 primary antibody (PharMingen), while anti-rat Alexa Fluor Texas Red (Molecular Probe) was used as secondary antibody. Details are reported in (Supplementary Information).

For immunohistochemical analyses, frozen cardiac tissues were embedded in OCT compound (Bio-Optica, Milan, Italy). Sections (7 µm) were incubated with mouse anti-Cx43 and rabbit anti-TOM20 for 2 h at room temperature. Then the slides were washed three times with PBS and incubated with secondary antibodies (FITC-conjugated anti mouse IgG and Texas red-conjugated anti rabbit IgG) for 1 h. DAPI was used to mark the nuclei. After mounting, coverslips were examined by using a Laser Confocal Microscope (Leica TCS SP5, Wetzlar, Germany).

GFP-labeled EpH4,EpXT, EpRAS treated and untreated with TGF-β1, EpXT-shTGFβ-RII cells were grown in culture flasks, trypsinized, washed and resuspended in phosphate-buffered saline (PBS), and injected at a total number of 1 × 10⁶ cells into the right hind footpads of 8-week-old BALB/c mice. After 1, 2 or 6 days, footpad tissues from the injection sites, and both ipsilateral and contralateral PLN, were isolated and fixed with 4% paraformaldehyde in PBS for 3 h. Tissues were embedded in OCT compound (Bio-Optica, Milan, Italy) and frozen.
For CCR7 inhibition studies, GFP-labeled EpXT cells were transfected with siRNA against CCR7 and scramble control (Dharmacon/GE Healthcare/VWR International, Stockholm, Sweden) for 24 h. Next day, a number of 1 × 10⁶ of EpXT-siCCR7 or EpXT-sicontrol cells were injected into the right hind footpads of 8-week-old BALB/c mice.
For p38 inhibition studies, GFP-labeled EpXT cells were pretreated with 20 μM SB203580 or vehicle (dimethyl sulfoxide) for 48 h before footpad injections. A total number of 1 × 10⁶ GFP-labeled EpXT cells were resuspended in PBS containing 20 μM SB203580, or vehicle, and injected into the right hind footpad of 8-week-old BALB/c mice.