New Perspectives on Melanoma_ The Role of PAX3

A2aliquotofsheareddnapriorto immunoprecipitation was

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Unformatted text preview: RNA copy number for each sample relative to that in the normal skin was expressed as a fold‐change increase in mRNA. Antibodies The following primary antibodies were used: mouse monoclonal to PAX3 (DSHB, 1/10), rabbit polyclonal to PAX3 (Invitrogen, 1/500), mouse monoclonal to MITF (Merck, 1/20), mouse monoclonal to MLANA (Merck, 1/50), rabbit polyclonal to HES1 (Abcam, 1/100), rabbit monoclonal to BCL2L1 (Abcam, 1/50), rabbit monoclonal to MCAM (Abcam, 1/500) and rabbit polyclonal to Ki67 (Abcam, 1/25). For immunofluorescent staining the following secondary and tertiary antibodies were used: anti‐mouse conjugated AlexaFluor‐488 (1/500), anti‐rabbit conjugated AlexaFluor‐488 (1/500), anti‐mouse conjugated AlexaFluor‐546 (1/500), or biotinylated anti‐rabbit IgG (1/500) linked to streptavidin‐conjugated AlexaFluor‐546 (1/500). All antibodies were diluted in PBST (0.2% TritonX‐100 in PBS) together with 1%NGS for immunofluorescent staining. Immunohistochemistry Paraffin embedded sections were dewaxed in xylene (3x 5 minutes) and dehydrated in ethanol series (3x 5 minutes in 100% ethanol, 1 minute in each of 95% and 70% ethanol, and 3x 10 dips in ddH20). Antigen retrieval was routinely performed in EDTA/Tris, pH 8.0, by heating for 3x 5 minutes in a microwave oven, at approximately 750 Watts. Slides were left to cool to room temperature for at least 20 minutes. Sections were washed in PBS and endogenous peroxidases were blocked with 3% H202 for 10 minutes. After rinsing in PBS, sections were blocked with 10% FCS for one hour, followed by incubation with primary antibodies for one hour at room temperature. After washing in PBS, the secondary antibody 74 Chapter III – Medic and Ziman, 2010 linked to biotin (Dako, LSAB kit) was applied and sections were incubated for 20 minutes at room temperature. After washing in PBS‐Tween20 (0.05%), sections were incubated with streptavidin conjugated horseradish peroxidase (Dako, LSAB kit) for 20 minutes at room temperature, washed in PBS‐Tween20, then incubated with DAB (Da...
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