Histological Analysis of Muscle Tissue

The frozen tissues were fixed in paraformaldehyde (4% w/v) and transferred into ethanol (70% w/v). The samples were then paraffin embedded and sectioned using a microtome. Muscle sections were then stained with Hematoxylin and Eosin (H&E) stain for anatomical features.^{[9 (link)]} The samples were also immunostained using antibodies staining for von Willebrand factor (1:1000; DAKO), CD86 (1:250; Bioss) and CD163 (1:100; Bioss). The staining was detected using DAKO Envision kit using a 3,3′-di-amino benzidine (DAB) substrate. The vessel density was quantified by manually measuring the positively stained cells on Metamorph (Molecular Devices). Cells positive for CD86 or CD163 positive cells were quantified using the online tool “ImmunoRatio,” an automated image analysis software for DAB immunostained images.^{[16 (link)]}