*2.6. Histological and Immunohistochemical Study*

A skin flap was excised from the affected area (the area of the wound defect, with the adjacent skin and underlying muscles) and fixed in a 10% solution of neutral formalin. Further processing of excised samples was performed using standard histological methods. Preparations stained with hematoxylin and eosin were used for general assessment of the condition of the studied tissues.

Immunohistochemical examination of tissue samples on days 28 and 112 of the experiment was performed using an automated method, a Ventana BenchMark Ultra immunostainer with dewaxing and unmasking in an apparatus using antibodies to VEGF (Novocastra, Milton Keynes, UK, skin blood vessel endothelium marker), CD31 (Novocastra, endothelial cell marker), CD68 (Novocastra, macrophage marker), PGP9.5 (Novocastra, marker of differentiating neurons in the skin), Ki67 (Novocastra, marker of cell proliferation), FVIII (Novocastra, marker for platelet adhesion factor), collagens of types I and III (Novocastra), tissue inhibitor of metalloproteinases TIMP2 (Novocastra), and metalloproteinases of types 2 and 9 (Novocastra). Marker expression was evaluated semiquantitatively, assigning scores from 0 to 3, where 0 means no expression and 3 means fully expressed.

Statistical analysis of the results was performed using Microsoft Office Excel 2007 (Redmond, WA, USA), Statistica 6 (Round Rock, TX, USA) and ImageTool software (San Antonio, TX, USA. The Mann-Whitney test was used to assess the significance of the differences.
