The vital dye Aniline Blue diammonium salt (Sigma-Aldrich 415049) dissolved in 1 M Glycine (pH = 9.5) (0.1% working solution) was adopted for PD-associated callose staining upon stoma infiltration with a needleless syringe into Nicotiana tabacum leaves for confocal microscopy analyses. Staining was performed three times in three independent lines with consistent results.
Aniline blue diammonium salt
Manufactured by Merck Group
Sourced in United States, Spain
Aniline Blue diammonium salt is a synthetic dye commonly used as a staining agent in various laboratory applications. It has a chemical formula of (C₆H₅NH₂)₂·H₂SO₄ and appears as a dark blue crystalline powder. The dye is soluble in water and exhibits a vivid blue color when dissolved. Aniline Blue diammonium salt is widely employed in staining techniques, particularly in the field of microscopy, to enhance the visualization and contrast of biological samples.
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Lab products found in correlation
Entellan, by Merck Group (1 mentions)
Absolute ethanol, by ITW Reagents (1 mentions)
Hydrochloride acid, by Merck Group (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
Phenol, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Sodium hydroxide pellet, by Merck Group (1 mentions)
4 protocols using aniline blue diammonium salt
1
Callose Staining in Tobacco Leaves
2
Tissue Sectioning and Staining for Confocal Microscopy
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Stromata (STR) and substromal stem (SUB) samples were cut in approximately 0.5 cm cross sections by hand with a scalpel blade. The samples with wheat germ agglutinin-Alexa Fluor 488 Conjugate (ThermoFisher Scientific, Massachusetts, USA) and Aniline Blue diammonium salt (Sigma-Aldrich Chemie GmbH, Traufkirchen, Germany) as previously described (Becker et al. 2018 ). Sections of 100 µm were taken with a vibration microtome (Thermo Scientific, Microm HM 650V) cutting an approximately 1 cm3 block of 5% agarose (Agarose SERVA Low Melting; SERVA, Heidelberg, Germany) containing the pre-stained samples. The 100 µm slices were transferred to a slide and embedded in staining solution for 10 min before confocal laser-scanning microscopy was performed as previously described (Becker et al. 2018 ).
3
Triple Staining Technique for Histology
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A triple staining with Aniline blue, acid Fuchsin and Orange-G (AFOG) was performed as previously described [7 (link)]. For the single-dye staining with Aniline blue, cryosections were refixed with 10% neutral buffered formalin solution for 15 min and washed in PBST for 10 min. The slides were transferred into preheated Bouins fixative (Lot. 1536576, Reactorlab) and incubated for 2.5 hours at 56°C and another hour at room temperature. The slides were washed twice for 20 minutes in tap water and then incubated in 1% phosphomolybdic acid for 5 minutes. After rinsing with distilled water, sections were incubated for 4 minutes with aniline blue staining solution (1 g aniline blue diammonium salt, Sigma Aldrich, 415049; 200 ml acidified distilled water) and again washed with distilled water. The sections were dehydrated in 95% ethanol and twice in 100% ethanol for 2 minutes. After passing through three consecutive xylol baths, the slides were mounted with Entellan (107961, Merck Millipore).
4
Characterization of Lentinula edodes Glucans
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Powdered Lentinula edodes S. (Berkeley) fruiting bodies with a particle size lower than 0.5 mm and a moisture content less than 5% were purchased from Glucanfeed S.L. (La Rioja, Spain). Absolute ethanol was purchased from Panreac (Barcelona, Spain) as well as concentrated sulfuric acid. Phenol, sodium borohydride, sodium hydroxide pellets, glycine, aniline blue diammonium salt 95% and hydrochloride acid 37% were obtained from Sigma-Aldrich (Madrid, Spain). Compounds used as standards were two linear β-D-glucans: curdlan (a (1→3)-β-D-glucan from Alcaligenes faecalis, Sigma-Aldrich, Madrid, Spain) and a (1→6)-β-D-glucan isolated from A. bisporus (named B6G) (Smiderle et al., 2013) ; and two branched (1→3),(1→6)-β-D-glucans:
schizophyllan from Contipro Biotech (Dolni Dobrouc, Czech Republic) and a chemically characterized glucan (PUL, previously named B1316PP) extracted from P. pulmonarius (Smiderle et al., 2008) . Moreover, a heteropolysacharide (mannogalactan, MG) isolated from P.
pulmonarius (Smiderle et al., 2008) and commonly found in Basidiomycetes, was also used to compare with the β-D-glucans.
schizophyllan from Contipro Biotech (Dolni Dobrouc, Czech Republic) and a chemically characterized glucan (PUL, previously named B1316PP) extracted from P. pulmonarius (Smiderle et al., 2008) . Moreover, a heteropolysacharide (mannogalactan, MG) isolated from P.
pulmonarius (Smiderle et al., 2008) and commonly found in Basidiomycetes, was also used to compare with the β-D-glucans.
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