Publication protocol
In IHC with enzyme detection, the sections were first treated with 3% hydrogen peroxide in methanol to inactivate intrinsic peroxidase activity, and then with 5% normal goat serum to prevent non-specific antibody binding. They were then incubated overnight at 4C with a mouse monoclonal anti-BrdU antibody (Clone Bu20a; DakoCytomation, Glostrup, Denmark; Magaud et al. 1989) at 1:150 dilution. After washing with PBS, the site of the immunoreaction was visualized by incubating the sections successively with a biotinylated anti-mouse IgG antibody (1:250; Vector Laboratories, Burlingame, CA) for 1 hr, horseradish peroxidase-conjugated streptavidin (1:300; DakoCytomation) for 1 hr, and ImmPact DAB (Vector Laboratories) for 1 min. Normal mouse IgG (10 μg/ml; Vector Laboratories) was used instead of the anti-BrdU antibody as a negative control. In IHC with fluorescence detection, the sections were treated with 1% bovine serum albumin alone and then incubated overnight at 4C with the mouse monoclonal anti-BrdU antibody (1:150), either alone for single immunofluorescence or in combination with a goat polyclonal anti-rat GFRA1 antibody (1:200; Neuromics, Edina, MN) (Naughton et al. 2006; Grasso et al. 2012), rabbit polyclonal anti-mouse PLZF antibody (1: 200, Sigma-Aldrich) (Meistrich and Hess 2013), rabbit polyclonal anti-mouse CADM1 antibody developed in our laboratory (0.5 μg/ml) (Wakayama et al. 2003, 2007; Nakata et al. 2012), or goat anti-mouse Gata4 antibody (1: 400; Santa Cruz Biotechnology, Dallas, TX) (Imai et al. 2004) for double immunofluorescence microscopy. After washing, the immunoreactive sites were visualized by incubating the sections for 1 hr at room temperature with the anti-mouse IgG antibody conjugated with Alexa Fluor 594 (1:400; Molecular Probes, Eugene, OR) alone for single immunofluorescence or in combination with the anti-rabbit or anti-goat IgG antibodies conjugated with Alexa Fluor 488 (1:400; Molecular Probes) for double immunofluorescence.
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