For frozen sections the tissue was cryoproserved in OCT and sectioned at 5 μm thickness. Horizontal wholemounts were prepared as described previously (Driskell et al., 2012 (link); Driskell et al., 2013 (link)). The following antibodies were used: Prominin-1 (1:50) (eBioscience #14-1331-82) Keratin 14 (1:1000) (Covance #PRB-155P) Corin (1:100) (R&D systems #AF2209) β-catenin (1:500) (Cell Signalling #8814S) and Ki67 (1:200) (Dako #M7249 clone TEC-3). All microscopy was performed on a Leica SP5 or Nikon A1 confocal microscope and images were analysed using Image J (NIH).
Protocol detail
Immunofluorescence Analysis of Skin Samples
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Antibodies
Clone
Confocal microscope
Frozen sections
Keratin 14
Microscopy
Prominin 1
Tissue
β catenin
Corresponding Organization :
Other organizations : King's College London
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Variable analysis
independent variables
- Tissue preparation method (cryoproservation in OCT and sectioning at 5 μm thickness, horizontal wholemounts)
- Expression of Prominin-1, Keratin 14, Corin, β-catenin, and Ki67
- Antibody concentrations (1:50, 1:1000, 1:100, 1:500, 1:200)
- Microscopy equipment (Leica SP5 or Nikon A1 confocal microscope)
- Image analysis software (ImageJ)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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