Callose deposits were stained as described previously.41 (link) Roots or shoots of 10-d-old seedlings grown on ½MS plate were dip-incubated with or without 1 µM Pep1 for 24 hours, followed by fixation in a 3:1 ethanol:acetic acid solution for 6 hours. The fixative was changed three times to ensure both thorough fixing and clearing of the tissues, which is essential for good callose detection in the roots. Seedlings were rehydrated in 70% ethanol for 2 hours, 50% ethanol for an additional 2 hours, and water overnight. After two washes with water, seedlings were treated with 10% NaOH for 10 minutes to make the tissues transparent. After three washes with water and one wash with 150 mM K2HPO4 (pH 9.5), seedlings were incubated in aniline blue staining solution (150 mM K2HPO4, pH 9.5, 0.01% aniline blue (Sigma-Aldrich)) for 1 hour and then washed with 150 mM K2HPO4 (pH 9.5). The seedlings were mounted on slides, and callose deposits were observed immediately using the Olympu BX53 microscope equipped with DP74 CCD camera under UV (excitation, 390 nm; emission, 460 nm).
Aniline blue
Manufactured by Merck Group
Sourced in United States, Germany
Aniline blue is a synthetic dye used as a staining agent in various laboratory applications. It is a deep blue or purple colored powder that is soluble in water. Aniline blue is commonly used to stain nucleic acids, carbohydrates, and other biological structures for visualization and analysis purposes.
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Lab products found in correlation
Bouin s solution, by Merck Group (8 mentions)
Biebrich scarlet acid fuchsin, by Merck Group (6 mentions)
Calcofluor white, by Merck Group (5 mentions)
Phosphomolybdic phosphotungstic acid solution, by Merck Group (5 mentions)
Acid fuchsin, by Merck Group (4 mentions)
Orange g, by Merck Group (4 mentions)
K2hpo4, by Merck Group (4 mentions)
Biebrich scarlet acid fuchsin solution, by Merck Group (4 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Eukitt, by Merck Group (3 mentions)
Weigert hematoxylin, by Merck Group (3 mentions)
Phosphomolybdic phosphotungstic acid, by Merck Group (3 mentions)
Preheated bouin s solution, by Merck Group (2 mentions)
Phosphomolybdic acid, by Merck Group (2 mentions)
Weigert s iron hematoxylin, by Merck Group (2 mentions)
Biebrich scarlet acid fusion, by Merck Group (2 mentions)
Ds oimc camera, by Nikon (2 mentions)
Eclipse 80i, by Nikon (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Weigert s hematoxylin, by Merck Group (2 mentions)
110 protocols using aniline blue
1
Visualization of Callose Deposition
2
Quantifying Callose-Mediated Fungal Resistance
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Seedlings were collected at three dpi and cleared for >24 hr in 100% ethanol. One day prior to analysis, samples were incubated for 30 min in 0.07 M phosphate buffer (pH 9), followed by 15 min incubation in a 4:1 mixture (v/v) of 0.05% w/v aniline blue (Sigma-Aldrich, 415049) in 0.07M phosphate buffer (pH 9) and 0.025% w/v calcofluor white (Fluorescent brightener 28, Sigma-Aldrich, F3543) in 0.1M Tris-HCL (pH 7.5). After initial staining, samples were incubated overnight in 0.5% w/v aniline blue (Sigma-Aldrich, 415049) in 0.07M phosphate buffer (pH 9) and scored with an epifluorescence microscope (Olympus BX 51) fitted with blue filter (XF02-2; excitation 330 nm, emission 400 nm). Germinated conidia (germ tubes) were divided between in two classes: non-arrested and arrested by callose. In each assay, 10 leaves from different plants for each (epi)genotype were analysed, amounting to >150 conidia-callose interactions. Statistically significant differences in resistance efficiency of callose (asterisks) were analysed using Pearson’s Chi-squared tests (p<0.05) in pairwise comparisons with Wt line (#602), using R (v3.5.1).
3
Aniline Blue Staining of Nicotiana benthamiana
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Aniline blue solution is prepared before use via mixing 0.1% Aniline blue (Sigma-Aldrich)-water solution with 1 M glycerol solution in a ratio of 2:3. The mixture was infiltrated into N. benthamiana leaves by using a 1 mL needle-free syringe. Thirty minutes later, Aniline blue fluorescence was observed under confocal microscope.
4
Aniline Blue Staining and Callose Quantification
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For aniline blue staining, seedlings were fixed and cleared in a solution of acetic acid/ethanol (1:3) overnight, followed by two washes of 30 min in 150 mM K2HPO4 and staining with 0.01% aniline blue (Sigma) for 2 h in the same buffer. Observations were performed under an epifluorescence microscope and images were captured with a NIKON microscopy camera coupled to the NIS software 4.45 (NIKON). Callose intensity quantification was performed according to Daudi et al. (2012) (link). Callose intensity was calculated with ImageJ by counting the number of callose spots and assigning a value from 1 to 10 (10, saturated signal; 9, over 250 spots; 8, between 200 and 249 spots; 7, between 150 and 199 spots; 5, between 100 and 149 spots; 3, between 50 and 99 spots; 2, between 5 and 49 spots; 1, between 0 and 5 spots). Average callose measurements were based on at least 20 leaf pictures taken from 12 different seedlings. Trypan blue staining for cell death visualization was performed as previously described (Alcázar et al., 2009 (link)).
5
Visualizing Transient Callose Cell Wall
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To visualize the transient CCW, EBP grains were extracted from flower buds with size of 15–18 mm (plants grown at 20–25 °C) or 32–35 mm (plants grown at 10 °C). The CCW was stained by decolorized aniline blue solution (0.1% (w/v) aniline blue (Sigma) in 0.1 M K3PO4 (pH 11.0)) at room temperature for 10 min. Stained samples were imaged by confocal laser-scanning microscopy (TCS SP8; Leica) using an argon laser for excitation at 405 nm, and a 494–544 nm filter for detection of fluorescence emission. Z-stack imaging was performed with a Leica TCS SP8 using the Galvo flow system and optimized settings with a step size of ~0.34 µm.
6
In vitro Synthesis of β-1,3-Glucan
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For in vitro β-1,3-glucan synthesis, the enzyme purified in detergent GDN (FKS1 or the FKS1(S643P) mutant; 0.02 mg ml−1) and donor UDP-Glc (2.5 mM) were mixed in reaction buffer (50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 6 μg ml−1 Rho1, 0.2% CHAPS, 0.04% CHS, 4 µM GTPγS and 20 mM KF), in the presence or absence of 200 μM caspofungin. The reaction volume was set up as 100 μl (for aniline blue staining) or 10 ml (for product enrichment and subsequent enzymatic degradation and glycosyl linkage analysis). The reaction was carried out at 30 °C for indicated time period.
For the staining of the synthesized products by aniline blue, a 100 μl aliquot of reactants or 0.1% (w/v) of S. cerevisiae β-glucan (Sigma) was taken and added to equal volume of aniline blue (0.03%; Sigma). The mixture was incubated in the dark for 20 min for complete dye binding. Each sample was then loaded into a capillary and was observed under a fluorescence microscope with an excitation wavelength of 365 nm and an emission wavelength of 433 nm. Each assay was repeated three times with similar results.
For the staining of the synthesized products by aniline blue, a 100 μl aliquot of reactants or 0.1% (w/v) of S. cerevisiae β-glucan (Sigma) was taken and added to equal volume of aniline blue (0.03%; Sigma). The mixture was incubated in the dark for 20 min for complete dye binding. Each sample was then loaded into a capillary and was observed under a fluorescence microscope with an excitation wavelength of 365 nm and an emission wavelength of 433 nm. Each assay was repeated three times with similar results.
7
Visualizing Pd-located Callose in N. benthamiana
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To visualize Pd-located callose, N. benthamiana leaves transiently expressing 35S-NbRGPs, as well as control leaves, were infiltrated with aniline blue solution (0.1% aniline blue (Sigma Aldrich, Burlington, VT, USA) in 0.01 M K3PO4 at pH 12. Then, the leaves were incubated in the dark at room temperature for 15 min before imaging using a Nikon C2 laser scanning confocal microscope. Quantification of the stained callose fluorescence was performed as described by Zavaliev and Epel [33 (link)].
8
Quantifying Callose Accumulation in Leaf Samples
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Leaf sections from uninfected and infected scions were stained overnight with aniline blue (Merck, Darmstadt, Germany) using a previously described method (Guenoune‐Gelbart et al., 2008 ). Stained leaf samples were then examined using a Zeiss (Oberkochen, Germany) LSM 780 laser confocal microscope with a 40× water immersion objective. Callose accumulation was detected at an excitation of 405 nm and emission between 413 and 563 nm was recorded. Callose quantification and deposit counting were performed as described by Zavaliev and Epel (2015 ) for eight replicate images per plant variety and treatment condition.
9
Nucleus Subcellular Colocalization and Callose Staining
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For the nucleus subcellular colocalization, the proteins were co-infiltrated with cultures (OD600 0.1) expressing the nuclear localization signal of SV40 large T antigen fused to the RFP, kindly provided by Dr. José Navarro IBMCP, Valencia, Spain.
As described by Leastro et al. (2018) (link) for callose staining, N. benthamiana leaves were infiltrated with aniline blue (Merck KGaA, Darmstadt, Germany) solution at 0.005% concentration in sodium phosphate buffer, 70 mM, pH 9.0. The leaves were infiltrated and kept in a dark room for 2 h before confocal visualization.
As described by Leastro et al. (2018) (link) for callose staining, N. benthamiana leaves were infiltrated with aniline blue (Merck KGaA, Darmstadt, Germany) solution at 0.005% concentration in sodium phosphate buffer, 70 mM, pH 9.0. The leaves were infiltrated and kept in a dark room for 2 h before confocal visualization.
10
Fluorescent Staining of Pollen Tubes
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For evaluation of fluorescent staining of pollen tubes on the medium and in the micropyle, aniline blue (415049; Merck, Germany), fluorescent brightener 28 (used as calcofluor white; F3543; Merck, Germany), Congo red (032-03922; Wako, Japan), PI (P4864; Merck, Germany), FM4-64 (F34653; Thermo Fisher Scientific, Waltham, MA, USA), and FDA (F7378; Merck, Germany) were prepared in the pollen germination medium without agarose. The final concentration of each dye is shown in Figure 1 . At 5.5 h after pollination, 20 µL of each dye was dropped on the medium. After 5 min, staining of pollen tubes was observed with an Axio Imager A2 upright microscope (Zeiss, Jena, Germany) equipped with a cooled charge-coupled device (CCD) camera (Axiocam 506 color; Zeiss, Germany). Filter set 47 HE was used for aniline blue and calcofluor white. Filter set 31 was used for Congo red, PI, and FM4-64. Filter sets 09 and 38 were used for FDA. To quantify pollen tube attraction into ovules, pollen tubes were stained with 5 µM FDA dissolved in hydrated silicone oil (KF-96–100CS; Shin-Etsu Chemical, Japan). After 5 min of staining, pollen tubes were observed.
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