Measurement of VEGF level
Measurement of VEGF levels in tissue samples was carried out with ELISA
kit [Sun Red, Shanghai Sunred Biological Technology Co., Ltd.].
First of all, standard samples were prepared. Standard prefixes of 50,
100, 200, 400, 800 and 1600 ng / L were prepared from the stock
solution. 0.05 ml standard and 0.05 ml HRP-streptavidin were added to
the wells determined in the ELISA plate. Samples were pipetted. The
plate was covered and incubated at 37 ° C for 90 minutes. For tissue
samples, 0.04 ml sample, 0.01 ml VEGF antibody and 0.05 ml
HRP-streptavidin were added. ELISA plate was incubated at 37 C for 60
minutes. At the end of the period, all samples were removed from the
ELISA plate with a pipette and washed 3 times with washing buffer. At
the end of the washing process, chromogen A [0.05 mL] and B
solutions [0.05 ml] were added to the wells and the plate was
incubated at 37 ° C for 15-30 minutes. At the end of the period, the
reaction was stopped by adding 0.05 ml of stop solution to the wells.
The optical density of the resulting yellow color was read in a
microplate reader [Thermo Scientific® Multiskan Go] at 450 nm
wavelength. Results were evaluated according to the standard calibration
curve.