Apo ciii

The 4-µm thick biopsy specimens were first deparaffinized in xylene I and II for 10 minutes each, rehydrated in ethanol I, ethanol II, 95% ethanol, and 80% ethanol for 5 minutes each, and rinsed with phosphate buffered saline (PBS, pH7.4) twice for 5 minutes each. Samples were then soaked in 3% hydrogen peroxide for 10 minutes, followed by a rinse with tap water. A SPlink Detection kit (ZSGC-BIO/ORIGENE, Shenzhen, China) was used for immunohistochemical staining following the manufacture's protocol. Briefly, samples were heated in 0.01 M sodium citrate (pH6.0) for 10 minutes at 95℃, followed by three rinses with PBS for 5 minutes each. Samples were incubated with primary antibodies (rabbit anti-ApoE, ApoC-III, and ApoA-1, diluted 100, 100, and 200 times, respectively; Abcam, Cambridge, United Kingdom) for 2–3 hours at room temperature, and then rinsed three times with PBS for 5 minutes each. Samples were incubated with secondary antibody (biotin-conjugated goat anti-rabbit IgG at a dilution of 1:400) for 20 minutes at room temperature, followed by three rinses with PBS for 5 minutes each. Samples were then stained with DAB kit (ZSGC-BIO/ORIGENE) following the manufacture's protocol and mounted with neutral resin on slides.