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.
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Immunohistochemistry Mouse - PLZF using Anti-ZBTB16 antibody produced in rabbit from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.