Fluorescent brightener m2r

In order to evaluate the localization of chitin in the demineralized skeleton of A. wolffgangi and E. gibbosa, Calcofluor White (Fluorescent Brightener M2R, Sigma) was used as a fluorescent dye for staining of β-(1→3) and β-(1→4) linked polysaccharides [31 (link),49 –52 (link)]. After binding to polysaccharides containing β-glycosidic bond, such as chitin, this flourochrome emits a bright blue light under UV excitation even using very short light exposure time (up to 1/1000 s) Selected fragments of demineralized skeletons of A. wolffgangi and E. gibbosa were placed in 0.1 M KOH-glycerine-water solution and few drops of 0.1% solutions of CFW were added and the mixture was placed in darkness for 60 min. Afterwards, the stained skeletons were rinsed 5 times with deionized water and dried at room temperature followed by investigation of the scaffolds under fluorescence microscopy.

The Calcofluor white staining (CFW) (Fluorescent Brightener M2R, Sigma-Aldrich, St. Louis, MO, USA) was used to confirm the presence of chitin in the sponge skeleton several times (see [5 (link),6 (link),7 (link),8 (link)]). For the staining process, 30 µL of a solution containing 10 g glycerin and 10 g NaOH in 90 mL of water was applied. After a minute, the CFW was added, and the investigated material was incubated in staining solution for 6 h without light at 25 °C. Then, the sample was washed with distilled water to eliminate the unattached stain, dried at 25 °C and analyzed using fluorescent microscopy. On binding to polysaccharides containing β-glycosidic bonds (such as chitin), this fluorochrome secretes bright blue light under UV excitation even with a very short light exposure time.

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.

Calcofluor white (CFW, Fluorescent Brightener M2R, Sigma-Aldrich, St. Louis, MO, USA) staining was applied preliminarily to confirm the presence of chitin in materials isolated by the electrolysis-assisted method. This technique was used several times in different variants [9 (link),14 (link),70 (link),74 (link)]. However, in this study, the 30 µL of a solution containing 10 g glycerin and 10 g NaOH in 90 mL of water was applied as a buffer. Subsequently, after 5 min, the CFW was added to the samples, and the investigated materials were incubated in staining mixture for 6 h without sunlight at 25 °C. After this treatment, samples were extracted from the staining solution and extensively rinsed with distilled water to eliminate the free unbounded molecules of CFW stain, then dried at 25 °C and investigated using fluorescent microscopy.