Aec solution

Paraffin-embedded samples were cut into 5 μm thick sections. Sections were deparaffined, rehydrated before antigen retrieval by 20-min incubation at 96°C in citrate buffer at pH 7.3 and nonspecific sites were blocked with 5% BSA. For immunohistochemistry (IHC), endogenous peroxidase was inhibited by incubation with 3% H₂O₂, the slides were then successively incubated with primary antibodies, biotin-conjugated secondary antibodies and HRP-conjugated streptavidin in PBS with 0.1% Tween and 1% BSA before incubation with AEC solution (Vector Laboratories) and counterstaining with hematoxylin. Apoptotic cells were detected by TUNEL assay using the ApopTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore). For immunohistofluorescence (IHF), the slides were successively incubated with primary antibodies and fluorochrome-conjugated secondary antibodies in PBS with 0.1% Tween and 1% BSA and were finally mounted using Prolong Gold (Invitrogen). IHC and IHF images were captured on a Nikon Eclipse E400 microscope at a 40x magnification in the visible or fluorescence mode, respectively.

PBS was placed on a chip fixed with HBs adr antigen at room temperature, a baseline spectrum was measured. To compare signal enhancement methods, mouse anti-HBs antibody (Hs41) in PBS (50 µL) was added for an antigen-antibody reaction on a chip for 10 min and the chip surface was washed with PBS 5 times. Fluorescence- and gold nanoparticle-conjugated antibodies were prepared using anti-mouse IgG antibody reacted with the HiLyte Fluor 555 Labeling kit–NH₂ (Dojindo Laboratories, Kumamoto, Japan) and the 80 nm NHS-activated Gold Nanoparticle Conjugation kit (Cytodiagnostics, Burlington, Canada), respectively. Fluorescence-conjugated antibody (50 µL; 10-fold dilution) or gold nanoparticle-conjugated antibody (50 µL; 15-fold dilution) was incubated on a chip for 5 min. After washing with PBS five times, changes in the spectrum dip were measured with a WM sensor. The signal enhancement method using a peroxidase reaction was also performed with EnVision+System–HRP (anti-mouse) (antibody conjugated with HRP; Dako North America, Carpinteria, CA) as reported previously with a minor modification [10 (link)]. Peroxidase-conjugated antibody (no dilution) was incubated on a chip for 5 min. After washing with PBS 5 times, the peroxidase reaction was employed with AEC solution (Vector Laboratories, Burlingame, CA) for 1 min. Changes in the spectrum dip were measured with a WM sensor.