Coronal cryosections (12 μm) were blocked for 1 hour in 2% donkey serum and 0.2% TritonX-100. Sections were incubated overnight at 4 °C with primary antibody against p65 (GTX102090; Genetex, Irvine CA) and double-labeled with primary antibody against rat endothelial cell antigen-1 (RECA-1) (MA1-81510; Thermo Fisher Scientific Inc.). Species appropriate Alexa Fluor 555- or fluorescein isothiocyanate-conjugated secondary antibodies were used for visualization. Sections were coverslipped with ProLong Gold antifade reagent (P36930 ; Thermo Fisher Scientific Inc.) containing the nuclear stain, DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride). Omission of primary antibody was used as a negative control.
Unbiased measurements of signal intensity within regions of interest (ROIs) were obtained using NIS-Elements AR software (Nikon Instruments, Melville, NY, USA), as described previously [28 (link)]. The area that was evaluated was a rectangle, 360 μm × 440 μm, positioned within the ACA-MCA watershed territory. Specific labeling for p65 within the ROI was defined as pixels with signal intensity >2.0× background. For nuclear translocation of p65, the ROI was defined by DAPI labeling within the same territory.
Unbiased measurements of signal intensity within regions of interest (ROIs) were obtained using NIS-Elements AR software (Nikon Instruments, Melville, NY, USA), as described previously [28 (link)]. The area that was evaluated was a rectangle, 360 μm × 440 μm, positioned within the ACA-MCA watershed territory. Specific labeling for p65 within the ROI was defined as pixels with signal intensity >2.0× background. For nuclear translocation of p65, the ROI was defined by DAPI labeling within the same territory.
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