Vectastain elite rabbit igg kit

A standard hematoxylin and eosin staining technique was used for histological analyses of paraffin-embedded (5-μm) skin sections. For immunohistochemistry, paraffin sections were deparaffinized with xylene and rehydrated with ethanol. Antigen unmasking was performed by boiling the slides in citrate-buffer (pH = 6, Dako, Santa Clara, CA) in a microwave for 5 minutes. After blocking the sections with 10% normal horse serum for 1 hour, sections were incubated with an unconjugated rabbit anti-human anti-HSV-1 serum (1:300; B01141, Dako) overnight at 4 °C. Subsequently, sections were incubated with a biotin-conjugated anti-rabbit IgG for 60 minutes at room temperature with the Elite rabbit IgG Vectastain kit (Vector Laboratories, Burlingame, CA). Biotinylated antibodies were detected with horseradish peroxidase-streptavidin, and staining was visualized with amino-ethyl-carbazole (Dako). Finally, sections were counterstained with hematoxylin (Merck, Darmstadt, Germany), mounted with Aquatex (Merck), and examined with a conventional bright field microscope (Olympus AX70; Olympus, Tokyo, Japan). Rabbit serum (Vector Laboratories) was used as isotype control.

Immunodetection of 4-HNE adducts was conducted as previously described (Roychowdhury et al., 2009b (link)). Liver tissue was formalin-fixed and paraffin-embedded, after which it was sectioned and mounted. Immunohistochemistry was then performed using rabbit antisera against 4-HNE (Alpha diagnostic 1:100). Following primary incubation, sections were conjugated using Vectastain Elite Rabbit IgG Kit (Vector Labs) and visualized using DAB substrate chromogen. Following development, slides were then counterstained using Gill’s hematoxylin (Vector labs). Images were acquired on a 20X objective by a blinded investigator.