2.3. Tissue and Blood Sampling

Heparinized blood gained by cardiac puncture was centrifuged (2.300g at 4 °C for 20 min) and plasma was stored at − 80 °C. Prior to harvesting superficial collateral arteries for qRT-PCR (quantitative real-time PCR), both hind limbs were perfused with latex flexible compound (Chicago Latex, Chicago, IL) via a catheter in the abdominal aorta. For each mouse two superficial collateral arteries per side were isolated, snap frozen in dry ice and stored at − 80 °C until further investigations.

For histological analysis of collateral arteries, mice were perfused with 20 ml adenosine buffer (1% adenosine (Sigma-Aldrich), 5% bovine serum albumin (BSA, Sigma-Aldrich) dissolved in phosphate buffered saline (PBS, PAN Biotech, Aidenbach, Germany), pH 7.4), to assure maximum vasodilatation (Chillo et al., 2016), followed by 20 ml 4% paraformaldehyde (PFA, Merck, Darmstadt, Germany) (for paraffin embedding) or 20 ml 3% PFA (for cryopreservation) in PBS, pH 7.4.

For histological analysis adductor muscles were rinsed in 4%PFA for 24 h and after paraffin embedding cut in 4 μm cross-sections. For immunofluorescence staining the samples were placed in 15% sucrose (Sigma-Aldrich), dissolved in PBS, for 4 h, followed by 30% sucrose, dissolved in PBS, overnight at 4 °C. Afterwards the adductor muscle tissue was cryopreserved in tissue tek (Sakura, Alphen aan den Rijn, The Netherlands) and cut in 6 μm cross-sections.