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.
Substrate kits
Manufactured by Vector Laboratories
Substrate kits from Vector Laboratories are a collection of reagents and buffers designed to facilitate various enzymatic and immunological assays. These kits provide the necessary substrates for the detection and quantification of specific target analytes in biological samples.
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Dual-label Immunohistochemistry and Immunofluorescence
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Dual-label Immunohistochemistry and Immunofluorescence
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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.
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