To visualize fungal proliferation in infected tissue, the area 1–3 cm below the injection site was excised at 2–7 dpi. Fungal hyphae, i.e. chitin, were stained with wheatgerm agglutinin (WGA) coupled to AlexaFluor488 (Invitrogen). Plant cell walls were stained with propidium iodide (Sigma‐Aldrich, St. Louis, MO, USA). Leaf samples were incubated in staining solution (1 µg/mL propidium iodide, 10 µg/mL WGA‐AF488) and observed with an LSM780 Axio Observer confocal laser scanning microscope (Zeiss, Jena, Germany) under the following conditions: WGA‐AF488, excitation at 488 nm and detection at 500–540 nm; propidium iodide, excitation at 561 nm and detection at 580–660 nm. For fluorescent protein detection, leaf samples were directly observed by confocal microscopy using the following conditions for mCherry: excitation at 561 nm and detection at 580–630 nm. For plasmolysis experiments, a 1 m mannitol solution was infiltrated into the leaves and observed 30 min later. All experiments were carried out at least three times. To measure appressoria formation and penetration efficiency, infected maize leaves were harvested 18–20 h post‐infection and stained with calcofluor white (10 ng/mL). Calcofluor white stains fungal hyphae only on the leaf surface as it cannot stain penetrated hyphae. The appressorial marker used has been described previously (Mendoza‐Mendoza et al., 2009 ).
Propidium iodide
Manufactured by Zeiss
Sourced in Germany
Propidium iodide is a fluorescent dye used in flow cytometry and microscopy applications. It binds to DNA and emits red fluorescence upon excitation, allowing for the detection and analysis of cellular DNA content.
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Lab products found in correlation
Propidium iodide, by Merck Group (5 mentions)
Propidium iodide, by Thermo Fisher Scientific (5 mentions)
Syto9, by Thermo Fisher Scientific (2 mentions)
Oil immersion objective, by Zeiss (2 mentions)
Confocal microscope, by Zeiss (2 mentions)
Zen software, by Zeiss (2 mentions)
Syto9, by Zeiss (2 mentions)
Lsm 510 duo confocal microscope, by Zeiss (2 mentions)
Dioc6 3, by Zeiss (2 mentions)
Propidium iodide solution, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Lsm780 axio observer confocal laser scanning microscope, by Zeiss (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Uvitex 2b, by Zeiss (1 mentions)
Uvitex 2b, by Polysciences (1 mentions)
Csu x1 spinning disk head, by Yokogawa (1 mentions)
Dioc6 3, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Zen lite software, by Zeiss (1 mentions)
Rolera em c2 camera, by Zeiss (1 mentions)
12 protocols using propidium iodide
1
Visualizing Fungal Proliferation in Infected Plant Tissue
2
Live/Dead Staining of S. aureus Biofilm
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The live dead staining of bacterial cells was done using standard protocol,29 (link) with variations. Briefly, the glass cover slip with pre-formed S. aureus biofilm was washed extensively with double distilled water, followed by washing with PBS, pH 7.0. The staining for viable cells on the cover slips was performed by the addition and 5 μM of Syto9 (ThermoFischer Scientific, USA), diluted in DMSO. The staining was done for 10 min in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable S. aureus cells, propidium iodide (ThermoFischer Scientific, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the washed and rinsed Syto9 stained cover slip. The staining was done for 10 min, followed by washing with 1× PBS and final rinsing with double distilled water. The imaging was done by Confocal microscope (Zeiss, Germany) at an excitation/emission of 483/503 nm for Syto9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Germany). The acquired image was processed by Zen software (Zeiss, Germany).
3
Root Anatomy Analysis via Cryo-Sectioning
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The fixed root tips were embedded with Tissue‐Tek® O.C.T.TM compound and solidified at −80 °C. Forty micrometre transverse sections were cut using a cryo‐section machine (Leica), followed by double staining with 1% (w/v) UVitex 2B (Polyscience) and 10mM propidium iodide (Sigma‐Aldrich), followed by a 2× rinse with deionized water after each staining. The sections were observed with a Zeiss LSM710 confocal microscope with a 405 nm laser line for UVitex 2B excitation and a 514 nm laser line for propidium iodide.
4
Hypocotyl Breakage Analysis Workflow
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Seedlings were scanned (HP Scanjet 8300) and germinated hypocotyl lengths were measured in ImageJ. For hypocotyl breakage experiments, three 6-day-old etiolated seedlings were arranged in parallel and clamped with small rubber-capped alligator clips at the tops and bottoms of the hypocotyls in a constant force extensometer (Durachko and Cosgrove, 2009 (link)). A 4 g counterweight (0.4 N force) was lowered gently by hand to allow the samples to become taut before releasing it. In most cases, breakage occurred almost immediately after the counterweight was released. To image breakage patterns using fluorescence microscopy, hypocotyls were stained with 10 µg/mL Propidium Iodide (PI; Life Technologies) and imaged on a Zeiss Axio Observer microscope with a Yokogawa CSU-X1 spinning disk head and a 20X 0.5 NA air objective using a 561 nm excitation laser and 617/73 nm emission filter. Different hypocotyl regions were defined by dividing the hypocotyls equally into thirds, and the breakage location was recorded for each hypocotyl. To image cell adhesion defects in seedlings overexpressing pectin-modifying enzymes, hypocotyls were stained with 10 µg/mL Propidium Iodide and imaged on a Zeiss Axio Observer microscope with a Yokogawa CSU-X1 spinning disk head and a 10X 0.3 NA air objective using a 561 nm excitation laser and 617/73 nm emission filter.
5
Confocal Microscopy of Cellular Scaffolds
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Confocal microscopy was used to visualize the cells. The cells seeded on scaffolds were fixed with methanol (−20 °C), rinsed with PBS and stained. 3,3’-Dihexyloxacarbocyanine iodide (DiOC6(3)) was used to visualize the cellular membranes (Life Technologies, Carlsbad, CA, USA; 1 μg/mL in PBS, 30 min) and propidium iodide (Sigma-Aldrich, MO, USA; 5 μL/mL, 10 min) to visualize the cell nuclei. Between the incubations, the samples were rinsed with PBS. A Zeiss LSM 510 DUO confocal microscope was used for imaging (λex maximum = 488 nm, λem maximum = 501 nm for DiOC6(3); λex maximum = 536 nm, λem maximum = 617 nm for propidium iodide).
6
C. glutamicum Viability Assay with Nisin, CTAB, and Corynaridin
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A fresh culture of C. glutamicum ATCC 13032 was washed once in phosphate-buffered saline (PBS), and bacteria were resuspended in saline (0.9% [wt/vol] NaCl) at an OD600 of 1. An 87.5-μL concentration of the cell suspension was mixed with nisin, cetyltrimethylammonium bromide (CTAB), H2O, or the RPC fraction containing corynaridin to the indicated concentrations and incubated for 10 to 30 min in the dark. Then, the bacteria were stained using 12.5 μL propidium iodide (25 μg/mL) (Invitrogen, Darmstadt, Germany) and again incubated for 15 min in the dark. Samples were imaged using a Axio Observer Z1 (Zeiss, Oberkochen, Germany) in bright-field and fluorescence mode with a filter set for propidium iodide (excitation at 575 to 625 nm, emission at 660 to 710 nm). Images were acquired with a 63× lens objective and analyzed using the Zen software (version 2.3 SP1; Zeiss).
7
Quantifying Haloarchaeal Viability via SMG
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The effect of SMG on viability of haloarchaea was studied using propidium iodide staining and visualization using fluorescence microscopy. propidium iodide stains the cells that exhibit damaged membrane red in color indicating non-viable, metabolically injured or necrotic cells. propidium iodide cannot penetrate intact membranes of live cells, hence viable cells don’t take up the red color (Stan-Lotter et al. 2002 (link)). Briefly, the cells exposed to SMG and NG were stained with propidium iodide solution (Sigma, Germany) and observed under Fluorescent microscope (AV 10-Zeiss, Germany) with Apotome and 400 X filter using the Axiovision software for imaging and the number of damaged cells (cells stained red with propidium iodide) were counted (Rieger et al. 2011 ). The percentage of viable cells was calculated as: % viability = (number of viable cells counted/total number of cells counted) × 100.
8
Live-Dead Bacterial Cell Staining
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The live–dead staining of bacterial cells was performed according to the protocol described by Manoharadas et al., 2021 [31 (link)]. Briefly, the staining for viable cells was performed by the addition of 5 μM of SYTO9 (ThermoFischer Scientific, Bedford, MA, USA), diluted in DMSO. The staining was performed for 10 m in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable cells, propidium iodide (ThermoFischer Scientific, Bedford, MA, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the cells. The staining was performed for 10 m, followed by washing with 1× PBS and final rinsing with double distilled water. The image was captured by confocal microscope (Zeiss, Jena, Germany) at an excitation/emission of 483/503 nm for SYTO9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Jena, Germany). The acquired image was processed by the Zen lite software (Zeiss, Jena, Germany).
9
GFP-RGA Localization in Arabidopsis Hypocotyl
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GFP-RGA protein localization in hypocotyl cells of Arabidopsis was investigated in 3-day-old pRGA::GFP-RGA etiolated seedlings grown on MS medium. Living hypocotyls were stained with propidium iodide (PI; Thermo Fisher Scientific, Waltham, MA, USA) before confocal imaging. The hypocotyl images were captured using Zeiss LSM880 confocal microscope with Airyscan detector, under 488 nm laser excitation and 509 nm emission for GFP channel and 586 nm laser excitation and 600 nm emission for the propidium iodide (PI) channel.
10
Phenotyping eif4e1 Mutant Embryos and Roots
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To identify the phenotype of embryos and RM of eif4e1-1 and eif4e1-2, the ovules and seedling roots were cleared in modified Hoyer's solution and an HCG solution as previously described (Chen et al., 2011 (link)). The samples were observed by using an Olympus BX51 microscope connected to a Ritiga 2000R camera. Seedlings were observed under a Nikon SMZ1000 microscope equipped with a Nikon digital sight Ds-Fi1 camera.
For confocal microscopy, seedling roots were stained with 100 μg/mL propidium iodide (Sigma-Aldrich) and mounted in 5% glycerol. Images were taken using a Zeiss LSM 780 confocal laser scanning microscope with the following excitation (Ex) and emission (Em) wavelengths (Ex/Em): 488 nm/505–530 nm for GFP, 561 nm/591–635 nm for propidium iodide, and 543 nm/600 nm for FM4-64 as previously described (Huang et al., 2014 (link)).
For confocal microscopy, seedling roots were stained with 100 μg/mL propidium iodide (Sigma-Aldrich) and mounted in 5% glycerol. Images were taken using a Zeiss LSM 780 confocal laser scanning microscope with the following excitation (Ex) and emission (Em) wavelengths (Ex/Em): 488 nm/505–530 nm for GFP, 561 nm/591–635 nm for propidium iodide, and 543 nm/600 nm for FM4-64 as previously described (Huang et al., 2014 (link)).
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