Rabbit anti 5 hydroxymethylcytosine

FFPE tissue slides were triple stained for 5-mC (mouce anti-5’-methylcytosine (Millipore, Temecula CA); Rabbit anti-5’-hydroxymethylcytosine (Active Motif, Carlsbad, CA).
The slides were examined with a Nikon 400 fluorescent microscope. The fluorescence intensity of staining of the protein of interest was measured quantitatively using 40× objective and a standard exposure time of 800 ms using a Nikon 400 fluorescent microscope with three filters (FITC-green, Texas Red, and Tri-Color), and the Nikon morphometric system. The results were displayed as a graph attached to the immunofluorescent photography using a screen snip.

Immunohistochemistry was performed on all cases (N=30). Sections were incubated overnight with a mixture of rabbit-anti-5-hydroxymethylcytosine (Active Motif, Carlsbad, CA) and mouse-anti-MART-1 antibodies (Covance, Princeton, New Jersey). The sections were washed and subsequently incubated with a mixture of secondary antibodies, including alkaline phosphatase-linked anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and peroxidase-linked anti-mouse IgG (Vector Laboratories, Burlingame, CA). The sections were then treated with their corresponding substrate kits (Vector Laboratories, Burlingame, CA). In addition, dual-labeling immunofluorescence was performed to complement immunohistochemistry as a means of two-channel identification of epitopes with nuclear staining of 5-hydroxymethylcytosine and membranous staining for MART-1. Instead of incubation with the secondary antibodies, these sections were incubated with a mixture of goat anti-rabbit IgG (Alexa Fluor, Grand Island, NY) and goat anti-mouse IgG (Alexa Fluor, Grand Island, NY). Appropriate isotype-matched antibody controls and tissue controls were employed for all experiments.