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.