Aniline blue solution

BAMs were fixed in 4% formaldehyde for 1 h and rinsed 3 × 5 min in PBS.
For tropomyosin staining, samples were permeabilized with methanol, incubated with mouse anti-tropomyosin antibody (Sigma #T9283) 1:100, followed by a secondary biotinylated anti-mouse IgG and then a preformed avidin and biotinylated horse radish peroxidase complex (Vector Laboratories, Burlingame, CA, United States). Development was by addition of DAB to produce a brown precipitate. Fixed BAMs were sent out for cross-sectional sectioning and hematoxylin and eosin staining (Mass Histology Service, Worcester, MA, United States). Slides were deparaffinized and then gradually rehydrated from 100% ethanol to H₂O and stained for collagen using Accustain Trichrome Stains (Masson) (Sigma-Aldrich). The Trichome protocol was adapted as follows. Slides were soaked overnight in Bouin’s solution (Sigma), stained in hematoxylin (Sigma), and then rinsed in running tap water. The slides were then placed in Biebrich scarlet acid fuchsin (Sigma), followed by 12 min in a working phosphotungstic/phosphomolybdic acid solution (Sigma), and then 6 min in an aniline blue Solution (Sigma). Sections were rinsed in double distilled water in between every staining step. The slides were then destained for 1 min using a 1% acetic acid solution (Sigma), dehydrated with ethanol, and mounted in Polymount (Polysciences Inc.).

The lung sections were stained with Ponceau (Sigma-Aldrich) and acid fuchsin for 5–10 min, treated with 1% phosphomolybdic acid for 5 min, and then counterstained with aniline blue solution (Sigma-Aldrich). All sections were subsequently dehydrated with ethanol, deparaffinized in xylene, and finally mounted with neutral balsam. For semiquantitative analysis of fibrosis by Ashcroft Score, four different levels separated by 100 μm were analyzed by two blinded independent investigators. Morphological changes in fibrotic lungs were quantified according to the criteria of Ashcroft et al. (4 (link)).

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.

This
staining technique was used to visualize collagen fibers. Paraffin-embedded
specimens were processed onto microscope slides as described in the
above section. The staining process involved a combination of Weigert’s
hematoxylin, 2.5% aqueous phosphomolybdic-phosphotungstic acid, Bouin’s
solution, working Biebrich Scarlet-acid fuchsin 1% aqueous solution,
and 2.5% aniline blue solution (Sigma-Aldrich), with more in depth
details having been previously reported.^{23 (link)}

After deparaffinization, the sample slices were dipped into Bouin’s solution (Sigma, St. Louis, MO, USA) and incubated for 1 h at 56 °C. Thereafter, they were washed thoroughly with de-ionized water for 10 min and dipped into scarlet solution (Sigma, St. Louis, MO, USA), phosphomolybdic–phosphotungstic acid (phosphomolybdic acid, Sigma, St. Louis, MO, USA; phosphotungstic acid, Sigma), and aniline blue solution (M5528-25G, Sigma, St. Louis, MO, USA) for 10 min each. The slices were also washed with DI water and dipped into acetic acid (Sigma, St. Louis, MO, USA) for 3 min and hematoxylin for 10 min. Finally, these slices were washed again with deionized water and dehydrated.

Masson’s trichrome staining was performed according to the manufacturer’s instructions (Polysciences, Warrington, USA). Briefly, after xylol cleaning and rehydration with reverse gradient alcohol, the slides were washed with distilled water. The slides were then stained with hematoxylin for 10 min and washed again in distilled water. Next, the sections were immersed in aniline blue solution (Sigma-Aldrich) for 10 min, rinsed in distilled water for 5 min, and washed again in distilled water. Finally, they were mounted using Entellan (Merck).