*4.6. Immunohistochemistry*

Mice were perfused with 10 mL vasodilation buffer (100 μg adenosine, 1 μg sodium nitroprusside, 0.05% BSA in PBS, pH 7.4) followed by 10 mL 4% PFA post mortem. Mm. adductores of ligated hind-limbs were harvested on day 3 after FAL and cryosectioned with a thickness of 10 μm. Immunostaining was performed using the following antibodies: RAT ANTI MOUSE CD68: Alexa Fluor 488 Antibody (AbD Serotec, Düsseldorf, Germany), CD163 (M-96) Antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Donkey anti-Rabbit IgG: Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA), DAPI. The two largest collateral vessels were selected. Immunopositive cells were classified as classically activated macrophages (CD68+/CD163−) or alternatively activated macrophages (CD68+/CD163+). Confocal imaging was carried out using a Leica SP5 (Wetzlar, Germany). Acquired images were processed with ImageJ Software (National Institutes of Health, Maryland, MD, USA) for further analysis.
