Anti-VEGF Immunohistochemistry [IHC] Staining
After the preparations were deparaffinized, they were rehydrated with
decreasing concentrations of ethanol [99.6%, 96%, 90%, 80%, 70%,
50%]. Each alcohol batch was held for 2 minutes. Hydrogen peroxide
[3%] [H2O2] was applied at room temperature for 5 minutes,
followed by 3 washes with PBS. For permeabilization, after incubating
with 0.1% Triton-X 100 for 10 minutes at room temperature, it was
washed again 3 times with PBS. After 20 minutes of application with
protein block, it was incubated with anti-VEGF primary antibodies at the
dilutions recommended by the company at + 4 ° C for 1 night without
washing. After washing with PBS 3 times the next day, secondary
antibodies biotin [30 minutes] and then streptavidin [30
minutes] were applied. Washing was done with PBS 3 times between two
applications and after the last application. The diaminobenzedine
[DAB] chromogen was applied for 3-6 minutes to ensure the visibility
of immunoreactivities. Afterwards, the preparations washed with
distilled water at least 3 times were counterstained with Mayer’s
hematoxylin for 1 minute, then washed again 3 times with distilled water
and covered with enthallen. Control staining was done to test whether
the immunoreactivities were specific. Experiments were studied in 3
repetitions independently from each other and each group was 3 samples
(16).