Fluorescence imaging system

Western blots of NT-3, BDNF, and NGF were studied according to the method described in [18] . The rat cortex was homogenated and the protein content of the supernatant was estimated with an absorbance of 560 nm by using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL, USA). After electrophoresis and protein transfer to a polyvinylidene fluoride membrane, the membrane was stained with primary and secondary antibodies. After these procedures were complete, the membrane was reacted in a chemiluminescence reagent (Western Lightning^TM, Chemiluminescence Reagent Plus, PerkinElmer Life Sciences, Inc., MA, USA). It was then exposed to a high-performance chemiluminescence film (Amersham Biosciences, England, UK) to measure the optical intensity.
A fluorescence imaging system (Vilber Lourmat, France) was used to quantify the immunoreactivity and western blots according to the method described in [19] (link). The background optical intensity of the area near the examined cortical area and the immunoreactive band was measured. The optical intensity ratio of the ischemic cortex to that of the sham operation was determined after subtracting the background intensity. The means ± SDs of these values were calculated and were statistically compared at each time point between the 2 groups. Eight rats were included at each time point for the analysis.

Polyacrylamide gel (12%) was prepared with 1×Tris-borate-EDTA buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). Each sample was prepared with 1×Tris-borate-EDTA buffer containing 12 mM Mg²⁺, and the concentration of each DNA strand was 2 μM. The sample solution was heated at 90 °C for 10 min and then annealed slowly to room temperature. 20 μL of each sample was mixed with 2 μL of Gel-Dye Super Buffer Mix before loading into the gel. The gel was run under a constant voltage of 100 V over a period of about 2.5 h. Photographs were taken under UV light by using a fluorescence imaging system (Vilber Lourmat, Marne laVallee, France).