*2.5. Immunohistochemistry*

Mast cells were visualized by staining using a naphthol AS-D chloroacetate esterase staining kit (#91C, Sigma-Aldrich, St. Louis, MO, USA) and counted manually. A mast cell was considered resting when all granula were maintained inside the cell, while mast cells were assessed as activated when granula were deposited in the tissue surrounding the mast cell (examples are shown in Figure 1A). Tissue size was quantified by the Leica image analysis system (Leica Ltd., Cambridge, UK). Para ffi n embedded adductor and soleus muscle were stained with alpha smooth muscle actin (aSMA) (1A4, DAKO, Glostrup, Denmark) to visualize smooth muscle cell positive collaterals. Soleus muscles were stained for capillaries using CD31 (Sc-1506, Santa Cruz, Dallas, TX, USA) and macrophages using MAC3 (550292, BD-Pharmingen, Franklin Lakes, NJ, USA) together with aSMA and DAPI (Sigma, Santa Clara, CA, USA) as nuclear staining. For each antibody, negative controls were included using specific isotype-matched antibodies. The Pannoramic MIDI digital slide scanner (3DHistech, Budapest, Hungary) was used to create high-resolution images of the muscles. Snapshots (9 representative images per muscle) were taken using the caseviewer software (3DHistech) with a 40× magnification. aSMA positive collaterals were analyzed by counting the number of collaterals and measuring the diameters of each collateral with a visible lumen to determine arteriogenesis. CD31 positive capillaries and the number of MAC3 positive macrophages were quantified. All quantifications were performed using FIJI Image J image analysis software (ImageJ, Bethesda, MD, USA).

**Figure 1.** Mast cells in calf muscles of patients with peripheral artery disease. ( **A**) Representative high-magnification image of a non-activated (left) and activated (right) mast cell, stained using a chloro-acetate esterase (CAE) staining and indicated by arrows. The non-activated mast cell shows the pink granula in the cytoplasm of the cell, whereas the pink granula are being released in the surroundings of the activated mast cell. (**B**) Quantification of the number of mast cells/mm<sup>2</sup> tissue, and the percentage of activated mast cells observed (*n* = 15). ( **C**) Overview of a chloro-acetate esterase (CAE) staining of muscle tissue showing mast cells in pink (indicate by arrows) in between muscle fibers. ( **D**) Representative overview images of mast cells surrounding microvessels (indicated by \*) in human calf muscle tissue.

#### *2.6. FACS Analysis*

Blood was collected at sacrifice, after which red blood cells were lysed using an erythrocyte lysis bu ffer (0.1 mM EDTA, 10 mM NaHCO3, 1 mM NH4Cl, pH = 7.2). Subsequently, white blood cells were stained with the antibodies for flow cytometric analysis. Inguinal lymph nodes were harvested from all mice and processed through a 70 μm cell strainer to acquire single cell suspensions. Subsequently, the cell suspensions were stained for flow cytometry. In approximation, 200,000 cells per sample were stained with antibodies against extracellular proteins at a concentration of 0.1 μg/sample for 30 min as described previously [20,21]. All flow cytometry experiments were executed on a FACS Canto II (BDBiosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (v10, BDBiosciences).

#### *2.7. Statistical Analysis*

Results are presented as mean ± standard error of the mean (SEM). A 2-tailed Student's t-test was used to compare individual groups. Non-Gaussian distributed data were analyzed using a 2-tailed Mann–Whitney U test. *p*-values < 0.05 were considered statistically significant and are indicated with \*; *p*-values < 0.01 and < 0.001 are indicated by \*\* and \*\*\*, respectively.
