Intact and regenerating skin samples from sea bream (0 h, 6 h, 1, 2, 3 and 4 days after scale removal) were fixed in 4% PFA, decalcified overnight in 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 8) and dehydrated in ethanol (70, 90 and 100%), saturated in xylene and impregnated and embedded in low melting point paraffin wax (Histosec, Merck). Serial 5 μm sections of skin were mounted on 3-aminopropyltriethoxysilane (APES) coated glass slides, dried overnight at 37 °C, cooled to room temperature and stored until required. Masson’s trichrome staining was used to distinguish between collagen rich and/or mineralized and non-mineralized tissue as previously described [8 (link)]. Stained sections were analysed using a microscope (Leica DM2000) coupled to a digital camera (Leica DFC480) linked to a computer for digital image analysis. Digital images were used to quantify the thickness of the epidermis, basement membrane and dermis as well as the number and diameter (20 vessels per section) of blood vessels in intact (N = 3, 1 section per fish) and regenerating (N = 3, 1 section per fish) skin using ImageJ v1.44o software [63 ].
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