Aniline blue

Calcoflour White (CFW, Sigma-Aldrich) and aniline blue (AB, Sigma-Aldrich) were used to stain P. brasiliensis yeast cells to evaluate the effect of copper depletion in the composition of the cell wall, since CFW and AB specifically binds chitin and glucan respectively. P. brasiliensis grew in copper depletion or in presence of this metal for 24 h. The cells were stained with the dyes described above and analyzed by fluorescence microscopy as already detailed (De Curcio et al., 2017 (link)). In synthesis, for analysis of chitin amount, the cells were fixed in 100% methanol at -80°C for 20 min. The cells were collected, stained with CFW (100 μg/mL in PBS 1 X) for 30 min and washed with PBS 1 X. For analyses of glucan, the cells of P. brasiliensis were incubated with aniline blue solution (Sigma) for 5 min and subsequently washed twice with PBS 1 X. Both cells stained with CFW and aniline blue were visualized in a fluorescence microscope (Zeiss Axiocam MRc-Scope A1). The minimum of 50 cells for each microscope slides, in triplicates, were used to evaluate fluorescence intensity of the cells. The software provided the fluorescence intensity (in pixels) and the standard error of each analysis. Statistical comparisons were performed using the Student’s t-test and p ≤ 0.05 were considered statistically significant.

Callose was localized in root semithin sections (0.5–2 μm) stained with 0.05% (w/v) aniline blue (Sigma, C.I 42725) in 0.07 M K₂HPO₄ buffer, pH 8.5. Sections remained in aniline blue solution during observation with a Zeiss Axioplan microscope (Zeiss Oberkochen, Germany) equipped with a micrometric scale, a differential interference contrast (DIC) system and an Axiocam MRc5 digital camera. Lignin was observed under the optical microscope after staining with 3% Phloroglucinol.