*4.7. Immunohistochemistry*

Samples of tissue were deparaffined, hydrated, and equilibrated in phosphate buffered saline (pH 7.4). Rabbit polyclonal anti-COL-I and anti-COL-III (Abcam, Cambridge, UK) were used as primary antibodies to study collagen type I and III. Samples were incubated with secondary antibodies, anti-rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St. Louis, MO, USA). For COL-I and COL-III, the alkaline phosphatase procedure was performed. COL-I and COL-III were conjugated with avidin-alkaline phosphatase (ExtrAvidin-Alkaline Phosphatase, Sigma-Aldrich, St. Louis, MO, USA). It was used for 60 min at room temperature (Dilution 1/200 in PBS) and developed with the alkaline chromogenic substrate for 15 min (controlling the appearance of marking under the microscope). The chromogenic substrate preparation was performed immediately before development by adding 10 mL of PBS (10 mg of α-naphthol AS-BI phosphate, 10 mg of Fast Red, and 100 μL of 0.1 M levamisole) [64]. Nuclei were counterstained with Carazzi's hematoxylin (15 min). A negative control of the technique was performed without the primary antibody. Samples were mounting in aqueous medium with Plasdone. Tissue sections were examined under a Zeiss Axiophot light microscope equipped with an AxioCam HRc (Carl Zeiss, Oberkochen, Germany). A digital camera was used for observations. In order to calculate the percentage of protein expression, the German semiquantitative scoring system considering the staining intensity and area extent was used [65]. Immunostaining in the tissue was assessed by two independent histologists.
