Fixed samples were thawed and rehydrated with PBS containing 50mM glycine (Sigma) for 10 minutes. Cells were permeabilized with 0.1% Triton X-100 and non-specific binding sites were blocked with 0.5% bovine serum albumin (BSA). Slides were incubated for 1 hour at 37°C with primary antibodies against one of the following targets: eNOS (1:100 dilution, BD Biosciences, San Diego, CA), phosphorylated eNOS at serine 1177 (1:200 dilution, Millipore, Billerica, MA) and threonine 495 (1:100 dilution, Cell Signaling, Danvers, MA), Akt (1:100 dilution, Cell Signaling), PKCβ (1:100 dilution, Abcam, Cambridge, MA), phosphatase and tensin homolog (PTEN) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), nitrotyrosine (1:1000 dilution, Millipore, Billerica, MA), ICAM (1:150 dilution, Santa Cruz Biotechnology), IκBα (dilution, Novus, Littleton, CO), or p65 (1:375 dilution, Novus). All cells were also stained with an anti-von Willebrand Factor (vWF) antibody (1:300 dilution, Dako, Carpinteria, CA) to aid in endothelial cell identification. After incubation with the primary antibodies, the slides were washed and incubated with corresponding Alexa Fluor-488 and Alexa Fluor-594 antibodies (1:200 dilution, Invitrogen, Carlsbad, CA) and mounted under glass coverslips with Vectashield containing DAPI for nuclear identification (Vector Laboratories, Burlingame, CA). For each batch of patient-derived cells, we stained a control slide of cultured HAECs,, maintained in EGM-2 Bullet Kit medium (Lonza) at 37°C with 5% CO2 and taken from a single index passage.
Slides were imaged with a fluorescence microscope (Nikon Eclipse TE2000-E) at 20x magnification and digital images of the cells were captured using a Photometric CoolSnap HQ2 Camera (Photometrics, Tucson, AZ). Exposure time was held constant and image intensity was corrected for background fluorescence. Fluorescent intensity was quantified by NIS Elements AR Software (Nikon Instruments Inc, Melville, NY). For each protein of interest, fluorescent intensity was quantified in 20 cells from each patient and averaged. To minimize batch-to-batch variability in staining intensity, fluorescence intensity for each patient sample was normalized to the intensity of HAEC staining performed simultaneously. Intensity is expressed in arbitrary units (au) calculated by dividing the average fluorescence intensity from the patient sample by the average fluorescence intensity of the HAEC sample and multiplying by 100. Intensity quantification was performed blinded to subject identity and diabetes status, and each batch included patients with and without diabetes.
Slides were imaged with a fluorescence microscope (Nikon Eclipse TE2000-E) at 20x magnification and digital images of the cells were captured using a Photometric CoolSnap HQ2 Camera (Photometrics, Tucson, AZ). Exposure time was held constant and image intensity was corrected for background fluorescence. Fluorescent intensity was quantified by NIS Elements AR Software (Nikon Instruments Inc, Melville, NY). For each protein of interest, fluorescent intensity was quantified in 20 cells from each patient and averaged. To minimize batch-to-batch variability in staining intensity, fluorescence intensity for each patient sample was normalized to the intensity of HAEC staining performed simultaneously. Intensity is expressed in arbitrary units (au) calculated by dividing the average fluorescence intensity from the patient sample by the average fluorescence intensity of the HAEC sample and multiplying by 100. Intensity quantification was performed blinded to subject identity and diabetes status, and each batch included patients with and without diabetes.
