Chromotrope 2r

Cryosectioned tissues were brought to room temperature and immersed in acidified Harris modified hematoxylin (Fisher Scientific) for 5 min. Sections were then washed with running tap water until the water ran clear. Slides were incubated in Gomori trichrome stain (12.8 mM Chromotrope 2R (Sigma), 3.7 mM Fast green FCF (Sigma), 2.1 mM phosphotungstic acid (Sigma), 0.2 M acetic acid (Fisher), deionized water, pH 3.4) for 20 min at room temperature. Excess stain was removed with two quick rinses in freshly made 0.2% acetic acid, and slides immediately placed in 95% ethanol. Tissues were then dehydrated in ascending concentrations of ethanol, cleared and mounted as described above.

Chromotrope 2R and 3-hydroxyanthranilic acid were supplied by Sigma-Aldrich Chemical Co. (Milwaukee WI, USA); Fe(NO₃)₃, methyl orange, safranin T, phenol red, and H₂O₂ (30% m/m) were purchased from Vetec (Rio de Janeiro, Brazil); FeSO₄ was obtained from Synth (São Paulo, Brazil). Other reagents were of analytical grade from several suppliers. All reagents were used without prior purification. Deionized water was used to prepare all solutions. Table 1 summarizes some of the features of the dyes evaluated herein.

Isolated lungs were fixed in zinc‐buffered formalin (Anatech Ltd., Battle Creek, MI) overnight at 4°C, then stored in 70% ethanol. Transverse sections from left upper and lower lobes were paraffin‐embedded and cut into 10 μm sections. Slides were dewaxed in xylene overnight and rehydrated in 100%, 70%, and 50% ethanol. Antigen retrieval was performed using Antigen Unmasking Solution (Vector, H‐3300, Burlingame, CA). Endogenous peroxidase activity was quenched with 3% H₂O₂ in cold methanol (−20°C) and slides were permeabilized in Trypsin (Invitrogen, Carlsbad, CA) for 10 mins at 37°C. DNA was denatured using 2N HCl (30 min) and slides were blocked in 10% normal goat serum (Vector S‐1000, Burlingame, CA). Airway nerves were labeled with pan‐neuronal marker mouse anti‐PGP9 primary antibody (1:250 dilution 4°C overnight; ABD Serotech, Oxford, UK). Goat anti‐mouse biotinylated secondary antibody (Invitrogen) was then applied (1:400 dilution at room temp for 2 hours; Vector, VA‐9200, Burlingame, CA) followed by incubation in Vectastain Elite ABC (Vector, PK‐6100, Burlingame, CA) for 30 mins at room temperature. Slides were developed with Vector SG (Vector). Eosinophils were stained with 1% Chromotrope 2R (Sigma, St. Louis, MO) for 1 min at room temperature. Slides were allowed to dry and mounted in Cytoseal 60 mounting medium (Richard‐Allan Scientific, San Diego, CA).

Fecal samples were stained with Weber’s chromotrope stain (chromotrope 2R [Sigma-Aldrich, St. Louis, MO, USA], and Fast Green [Sigma-Aldrich, St. Louis, MO, USA] and phosphotungstic acid [Sigma-Aldrich, St. Louis, MO, USA]) [15 (link)] and microscopically screened for microsporidia spores at a magnification of 1000× under a Leica DM750 microscope model ICC50 HD (Leica Microsystems, Heerbrugg, Switzerland). Samples with spore-compatible structures, ovoid and refractile structures stained pink-red, were considered positive.

Sections of the distal colon and jejunum were fixed overnight in paraformaldehyde 3.7 % (Sigma-Aldrich, Diegem, Belgium) and embedded in paraffin. Sections of 5 µm were cut with a microtome. Specific quantification of eosinophils was performed using Chromotrope 2R staining [16 (link),19 (link)]. Briefly, following deparaffinization with xylene and rehydration with serial ethanol dilutions, slides were immersed for 1.5 h in a solution containing Phenol (106.2 mM; Sigma-Aldrich, Diegem, Belgium) and Chromotrope 2R (21.3 mM; Sigma-Aldrich, Diegem, Belgium). A counterstaining was performed by the application of hematoxylin during 15 s on the sections. The number of positive cells was counted in 5 nonoverlapping high-power fields per slide, one slide per animal, and expressed as the number of chromotrope-2R-positive cells per area of lamina propria.