4.4. Calcofluor White Staining Test

Calcofluor white (CFW) (Fluorescent Brightener M2R, Sigma-Aldrich, Taufkirchen, Germany), which shows enhanced fluorescence when it binds to polysaccharides, especially chitin, was used. The fragments of natural and demineralized sponge skeleton samples were placed in 0.1 M Tris–HCl buffer (pH 8.5) for 30 min. Afterwards, the samples were stained using 0.1% CFW solution for 2 h in darkness, rinsed several times with demineralized water, dried at room temperature during 5 h and analyzed using fluorescent microscopy.