BrdU pulses in melanoma spheres were performed for 8 h (4 μg/ml). Spheres were plated on DMEM/Matrigel (1:40) (BD Biosciences) to allow attachment, fixed with 4% paraformaldehyde and processed as previously described.60 (link) For apoptosis, spheres were immunolabeled with a rabbit anti-cleaved Caspase3 (Cell Signaling Technologies, Danvers, MA, USA), followed by an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. For SOX2 immunofluorescence, spheres were immunolabeled with a rabbit anti-SOX2 antibody (AB5603; Millipore, Billerica, MA, USA) and with an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. Formalin-fixed paraffin-embedded sections were subjected to antigen retrieval (with citrate buffer pH 6.0), incubated with rabbit anti-SOX2 antibody (AB5603; Millipore) and rabbit anti-cleaved Caspase3 (Cell Signaling Technologies) and visualized using UltraVision Detection System (Lab Vision, Fremont, CA, USA) and diaminobenzidine (Dako, Carpinteria, CA, USA).
Protocol detail
Melanoma Spheres: BrdU, Apoptosis, and SOX2
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Anti antibody
Antigen
Apoptosis
Brdu
Buffer
Caspase3
Citrate
Fluorescein
Formalin
Immunofluorescence
Isothiocyanate
Matrigel
Melanoma
Paraffin
Paraformaldehyde
Pulses
Rabbit
Sox2
Vision
Corresponding Organization :
Other organizations : Tumour Institute of Tuscany, University of Florence
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Variable analysis
independent variables
- BrdU pulses (4 μg/ml)
- Plating spheres on DMEM/Matrigel (1:40)
- BrdU incorporation (proliferation)
- Cleaved Caspase3 (apoptosis)
- SOX2 expression
- Incubation time (8 h)
- Fixation with 4% paraformaldehyde
- Immunolabeling and visualization protocols
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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