Figure 5

Immunofluorescent staining for PKC-βII. PKC-βII and TRITC signals from immunofluorescent staining of mesenteric arteries. Microvessels with diameter of approximately 100 μm were fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. They were later deparaffinized in xylene and rehydrated in a mixture of ethanol and distilled water. Antigens were unmasked using sodium citrate (10 mmol/L, pH 6.0), followed by exposure to a microwave heat source. Samples were then incubated at room temperature for 60 minutes with anti-PKC-βII at 1:100. Sections were washed in PBS and incubated with swine anti-rabbit IgG-TRITC (1:50 in PBS) for 30 minutes at room temperature. Immunofluorescent staining of small mesenteric arteries displayed a strong signal for PKC-βII in DM rats (Figure 5C). In contrast, the TRITC signals of anti-PKC-βII antibodies were weak in the controls and DM+cur rat vessels (Figure 5 B and D, respectively). The negative control displayed a minimal detectable fluorescence when the secondary antibodies were used alone (Figure 5A).