Biotinylated goat anti rabbit

For identifying cellular localization of IFN-β, we used IFN-β/YFP reporter mice. Tissue sections (16 µm) were rinsed in PBS containing 0.2 % Triton X-100 (PBST). The sections were incubated in blocking solution containing PBST and 3 % BSA, and stained either with rabbit anti-GFP antibody (ab6556; Abcam), PE-conjugated rat anti-mouse CD45 (BD Biosciences), Cy3-conjugated anti-GFAP or anti-Mac-1/CD11b (MCA711, Serotec, Oxford, UK). After 3 washes in PBST, sections were incubated with biotinylated goat anti-rabbit (Abcam), Alexa-569 goat anti-rat or streptavidin-HRP. GFP signal was amplified with TSA fluorescein kits (PerkinElmer) according to the manufacturer’s instructions. Sections were incubated with DAPI and mounted using gelvatol [17 (link)].
Images were acquired using an Olympus DP71 digital camera mounted on an Olympus BX51 microscope (Olympus, Ballerup, Denmark) and with Olympus FV1000MPE Confocal and Multiphoton Laser Scanning Microscope, Danish Molecular Biomedical Imaging Center (DaMBIC), University of Southern Denmark. Images were combined using Adobe Photoshop CS3 (Adobe Systems Denmark A/S, Copenhagen, Denmark) to visualize double-labeled cells.

Prepared slides were deparaffinized as above. The presence of tumor necrosis factor alpha (TNFα) in the livers was determined using immunohistochemistry. Slides were submerged in pre-heated sodium citrate buffer (pH 6.0) and heated at 98°C for 20 minutes to retrieve antigens. Liver sections were then incubated with hydrogen peroxide, protein blocking, and streptavidin peroxidase solutions at ten minutes each. Following each incubation step, the sections underwent two washing cycles. Each cycle involved five minutes of gentle agitation in a solution of TBS (Tris-buffered saline, pH 7.0) with 0.025% Triton X-100. Liver sections were then incubated with primary and secondary antibodies i.e., rabbit polyclonal anti-TNFα (cat# ab6671, Abcam, USA) and biotinylated goat anti-rabbit (Abcam, USA). To visualize TNFα, DAB (3,3’-Diaminobenszidine) chromogen was used to produce a brown colored substance at the site of the primary antibody. For histopathology, slides were then counterstained with hematoxylin.