Bs 1 b4

Cryo cross-sections of gastrocnemius or soleus muscle were cut in a cryostat, fixed in 4% PFA/PBS 10 min, endogenous peroxidase was blocked with 3% H₂O₂, and washed in phosphate-buffered saline (PBS; pH 7.4) at room temperature. Sections were then incubated 30 min at 37°C with 40 μg/ml in PBS HRP-conjugated Isolectin B₄ of Bandeiraea simplicifolia (BSI- B₄) (Sigma-Aldrich Co. LLC, ST. Louis, USA). Afterwards sections were washed with PBS and incubated with DAB solution (Roche Diagnostics), rinsed with PBS, counterstained with Mayer's hematoxylin (Carl Roth GmbH, Karlsruhe) and cover-slipped in mounting medium for histology, DePeX (SERVA Electrophoresis GmbH, Heidelberg, Germany). Nuclei were counterstained with Mayer's hematoxylin (Carl Roth GmbH). Capillary with a diameter of ≤ 5μm were identified in cross-sections by brown staining and quantified using the software software ImageJ (Scion Image, National Institutes of Health, Bethesda, USA). The average capillary numerical density (number of capillaries/mm²) and capillary contact per muscle fiber (number of capillaries/fiber) was calculated for each muscle cross-section. To analyze neuromuscular junctions α-Bungarotoxin (α-BT) histochemistry was performed (Materials and methods A in S1 File).

After the fresh enucleated eyes were fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature, the anterior segments were cut off and the choroid complexes (RPE/choroid/sclera) were flat-mounted to be permeabilized in PBS containing 0.5% Triton X-100 and Isolectin B4 (BSI-B4, L2895, Sigma, United States; 1:200) and were incubated at 4°C overnight. Images were taken using a fluorescence microscope and loaded into the ImageJ software to measure the CNV area. In brief, a thorough evaluation of the outline of each laser burn was carried out for the pixels within the evaluated area to be measured.