Method of Staining
Immunohistochemical VEGF, PDGFR-β, Thrombospondin stains were performed with the streptovidin-avidin-biotin method. The preparations evaluated at 40 magnification under light microscopy with a hematologist and a pathologist were scored as 0, 1, 2, 3 as per the staining ratio of the cells. The sections were deparaffinized by holding in a 56oC incubator overnight. Deparaffinization was completed by keeping the sections taken in xylene for 15+15 (30) minutes after they were removed from the oven. Then the absolute alcohol was poured first, kept for 15 minutes in the second, and then the same procedure was performed with 96% ethyl alcohol and hydrated. Washed with distilled water for 5 minutes. The slides were placed in a microwave-resistant plastic bowl. 10% Citrat buffer solution was prepared and placed on them (10 cc citrat buffer is prepared with 90 cc distilled water). The microwave oven was run at maximum power (100%) for 10 minutes. At the end of the time, the power of the oven was reduced by 50% and operated for 5+5 minutes. After the preparations were taken out of the microwave oven, they were kept at room temperature for 20 minutes. Washed with distilled water. The sections were kept in a mixture of 3% HO (Hydrogen peroxide) for 20 minutes to perform peroxidase blockage. Then the sections were washed with distilled water. After sections were kept in Phosphate buffer saline (PBS) for 15 minutes, they were placed in an immunostaining container and protein blockade was performed for 15 minutes. After washing, VEGF, PDGFR, Thrombospondin stains were dropped and left for 2 hours incubation. After the preparations were taken into PBS and shaken, they were kept in PBS for 15 minutes for the second time. Goat-Anti-Polyvalent was dropped on the sections taken into the staining container and left for 20 minutes. Then, after the preparations were taken into PBS and shaken again, they were kept in PBS for 15 minutes for the second time. Streptovidin peroxidase was dropped on the sections taken into the staining container and left for 20 minutes. Tissues that were passed through PBS again were incubated with AEC chromogen for 20 minutes. The colored preparations were washed in distilled water and then kept in Mayer hematoxylin for 2 minutes. After washing with distilled water, soaked through ammonia water. After the sections were washed with distilled water again and dried, they were closed with a suitable covering medium. The preparations evaluated at 40 magnification under light microscopy with a hematologist and a pathologist were scored as 0, 1, 2, 3 as per the staining ratio of the cells.