Wga 488

The staining method was slightly modified from Song et al. (Song et al., 2019 (link)). The fresh maize root segments were placed in a centrifuge tube containing 10% KOH and heated at 96°C for 5 min. The roots were washed with sterile water three times to remove the remaining KOH and placed in a centrifuge tube containing 2% HCl at 96°C for 5 min. After washing several times with 0.2 M PBS (pH = 7.4), the roots were re-immersed in PBS and placed at room temperature for 3 h. The roots were then transferred to PBS containing 5 µg/mL WGA-488 (wheat germ agglutinin-Alexa fluor 488 conjugate, Invitrogen, W11261) and stained overnight at 4°C. The stained root segments were washed several times with PBS and then placed in 0.2 M PBS containing 10 µg/mL PI for 1 h at room temperature, followed by washing with PBS. Stained root segments were observed under a Nikon Eclipse Ti2 inverted fluorescence microscope (Shanghai Nikon Instruments Co., Ltd., China), with an excitation wavelength of 488 nm and an emission wavelength of 507 nm, and photographed with a monochrome microscope camera (Nikon DS-Ri2). The mycorrhizal colonization rate was calculated as described by Biermann et al. (Biermann and Linderman, 1981 (link)).

Cells were stained using the following fluorophore conjugated anti-mouse antibodies: WGA-488 (Invitrogen), Ly6G-FitC (clone 1A8, BioLegend), Ly6G-BV510 (clone 1A8, BioLegend), Ly6G-PE (clone 1A8, BioLegend), CD11c-BV605 (clone N418, BioLegend), F4/80-FitC (clone BM8, BioLegend), F4/80-PE (clone BM8, BioLegend), CD11b-PE (clone M1/70, BioLegend), Ly6C-PE Cy7 (clone HK1.4, BioLegend), CD63-APC (clone NVG-2, BioLegend), CD9-APC (clone MZ3, BioLegend), IL-1α-PE (clone ALF-161, BioLegend), and IL-1β-APC (clone NJTEN3, ThermoFisher Scientific). Antibodies were diluted in wash buffer (PBS with 1% BSA and 2 mM EDTA). Cells were stained for 20 minutes at 4°C, washed with wash buffer, fixed for 15 minutes in BD Biosciences cytofix/cytoperm, and permeabilization prior to intracellular staining. ACEA Novocyte was used for flow cytometry, and Novoexpress software was used for subsequent analysis. AMNIS ImageStream was used for imaging flow cytometry and the AMNIS IDEAS software was used to calculate the colocalization coefficient.

The tomato roots with F. mosseae were stained with 0.01% cotton blue (w/v) in lactic acid. The A. sinicus roots with G. margarita or R. irregularis were immersed in a 10% KOH (w/v) solution at 37 °C for one week, neutralized in 2% HCl (v/v), washed three times with sterile water and then stained with 0.05% Trypan blue (w/v) or 5.0 μg/mL wheat germ agglutinin 488 (WGA488; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, respectively. AM fungal structures were observed with the appropriate microscopy. AM fungal spores and the stained fungal tissues were observed with a light microscopy (Nikon Y-TV55). Fluorescent signals in AM fungal spores and mycorrhizal roots were examined using a fluorescence microscopy (Nikon Y-TV55). The fungal spores were captured by a stereomicroscope (Nikon DS-R12). A Zeiss 780 laser scanning confocal microscope equipped with × 63 water immersion objective was used for the detection of arbuscules within roots. The excitation/emission of WGA488 were 488 nm/519 nm, respectively.

Rps. palustris or Rhodoplanes cells were resuspended in PBS and incubated with Alexa Fluor 488-conjugated wheat-germ agglutinin (WGA-488) or concanavalin A (ConA) (Invitrogen), respectively, at a final concentration of 0.5 µg/ml for 5 min prior to fluorescence microscopy imaging.

Primary and upper secondary roots of pea infected or not by A. euteiches, were collected at 15 dpi for microscopic analysis. The primary root was placed directly on a holder and the secondary roots were embedded in 2,5% agarose and sliced using a vibrating-blade microtome (Leica VT1000 S) to 100 μm thickness. To specifically stain A. euteiches hyphae, wheat germ agglutinin coupled to Alexa Fluor 488 conjugate (WGA-488, Invitrogen) was used. Briefly, the specimens were stained in a 10 μg/ml staining solution for 5 min at room temperature and directly placed in a water drop on a microscope slide and observed using a confocal microscope. A confocal laser scanning microscope (Leica TCS SP8 operated on the LAS X software platform) was used to image the samples. Alexa Fluor 488 was detected between 500-565 nm using an OPSL 488 nm laser. Specimens were observed using a 10X dry objective (HC PL FLUOTAR 10x/0.30). All images were processed using ImageJ software version 1.53.