Five-micrometer serial sections from each block were obtained in a micrometer (Leitz, 1512) and they were mounted on plus slides (Dako, S2024). All sections were attached to the slide by heating in a 60°C oven for 1 h. Afterward, the slides were dewaxed in xylene and rehydrated in graded ethanol series in the usual way. The samples were maintained in tap water until they were stained.
The immunolocalization of NeuGcGM3 ganglioside was performed as it was previously described in [10 (link)] with some modifications. Briefly, the slides were incubated with 14F7 Mab in a humid chamber for 1 h at room temperature followed by the labeled streptavidin biotin (LSAB) two steps' system (Dako, K0690) both for 30 minutes at room temperature. The enzymatic activity was visualized with 3,3-diaminobenzidine (DAB) substrate chromogenic solution (Dako, K3465) and the tissues were counterstained with Mayer's Hematoxylin (Dako, S2020). Concerning the evaluation of both EGFR and EGF tissue antigens, the procedure as it was previously described in [19 (link)] was used.
The immunolocalization of NeuGcGM3 ganglioside was performed as it was previously described in [10 (link)] with some modifications. Briefly, the slides were incubated with 14F7 Mab in a humid chamber for 1 h at room temperature followed by the labeled streptavidin biotin (LSAB) two steps' system (Dako, K0690) both for 30 minutes at room temperature. The enzymatic activity was visualized with 3,3-diaminobenzidine (DAB) substrate chromogenic solution (Dako, K3465) and the tissues were counterstained with Mayer's Hematoxylin (Dako, S2020). Concerning the evaluation of both EGFR and EGF tissue antigens, the procedure as it was previously described in [19 (link)] was used.
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