To determine the subcellular localization of MdTLP7, the MdTLP7 ORF without a termination codon was inserted upstream of the GFP gene. The 35S-GFP plasmid was used as a control. The subcellular localization experiment was carried out by Agrobacterium tumefaciens infiltration into the leaves of tobacco as described by Jia et al. [30 (link)]. After 48 h of infiltration, a two photon laser confocal microscope (ZEISS, Germany) was used to observe the fluorescence in tobacco cells. Fluorescence was detected using a 505 to 550 nm bandpass filter for GFP. Image processing was performed with the Zeiss LSM image processing software (Zeiss).
Lsm image processing software
Manufactured by Zeiss
Sourced in Germany
The LSM image processing software is a tool designed for the analysis and visualization of microscopic images. It provides a range of functionalities for image processing, including image segmentation, particle analysis, and 3D reconstruction. The software is intended for use with Zeiss microscopy equipment, but may be compatible with other imaging systems as well.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 airyscan, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Two photon laser confocal microscope, by Zeiss (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Lsm880 confocal microscope system, by Zeiss (1 mentions)
Lsm 900 laser scanning microscope, by Zeiss (1 mentions)
Tcs sp5ii, by Leica (1 mentions)
8 protocols using lsm image processing software
1
Subcellular Localization of MdTLP7 Protein
2
Microscopic Visualization of Plant Cell Structures
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FM4-64 staining of root epidermal cells [13 (link), 61 (link), 66 (link), 67 (link)] and Lysotracker red-staining of ovules [39 (link), 59 (link)] were as described. Fluorescent images were captured using a Zeiss LSM 880 confocal laser scanning microscope (CLSM) with a 40/1.3 oil objective. GFP-RFP double-labeled materials were captured alternately using line-switching with the multi-track function (488-nm for GFP and 561 nm for RFP). Fluorescence was detected using a 505- to 550- nm filter for GFP or a 575- to 650-nm band-pass filter for RFP. YFP-RFP double-labeled materials were captured alternately using line-switching with the multi-track function (514 nm for YFP and 561 nm for RFP). Fluorescence was detected using a 520- to 550-nm band-pass filter for YFP or a 575- to 650-nm band-pass filter for RFP. Differential interference contrast (DIC) imaging of ovules were performed using a Zeiss Axiophot microscope with DIC optics. Image processing was performed with the Zeiss LSM image processing software (Zeiss).
3
Quantifying VAC14-GFP Intensity in Guard Cells
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Fluorescence microscopy was performed by using a Zeiss LSM880 (Zeiss) confocal laser-scanning microscope with the following excitation/emission settings: 488 nm/505–550 nm for GFP or OG staining as described (Li et al., 2018 (link); Song et al., 2018 (link)). Images were processed using Zeiss LSM image processing software (Zeiss) and Adobe Photoshop CS3 (Adobe). For quantification of VAC14-GFP intensity, abaxial epidermal peels were taken from 3 WAG plants and placed in stomatal opening buffer. Fluorescence intensity was calculated as average intensity in guard cells subtracted by that in leaf pavement cells using ImageJ.
4
Subcellular Localization of TaPIN1 in Arabidopsis
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35S::TaPIN1-6a-CDS-GFP was inserted into the pMDC83 vector and infected into Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [33 (link)]. Staining of roots with FM4-64 was performed as previously described [34 (link)–39 (link)]. Confocal microscopy was performed on the root of the positive Arabidopsis plants with a Leica TCS SP5II (Richmond, IL, USA), and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880 Airyscan (Zeiss, German) with a 40 × objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software (Zeiss, German).
5
Immunoblotting and Confocal Microscopy
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Immunoblotting was performed using the method described by Zhang K. et al. (2014) . A Zeiss LSM880 confocal microscope was used to capture all fluorescence images using the following excitation or emission settings: 488 nm/505–550 nm for GFP and 561 nm/600–660 nm for mCherry. Images were processed using the LSM image processing software (Zeiss).
6
Fluorescent Protein Imaging of Arabidopsis
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10-DAG seedlings harboring pMDC163-SUC2pro-mCherry and pMDC107-AtABCG14pro-GFP or pMDC163-4CL1pro-mCherry and pMDC107-AtABCG14pro-GFP were observed under an LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany) using the following excitation or emission settings: 488 nm/505–550 nm for GFP and 561 nm/600–660 nm for mCherry. Images were processed using LSM image processing software (Carl Zeiss).
7
Confocal Imaging and Quantification of Root Meristem
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Root meristems were imaged by a Zeiss LSM 900 laser scanning microscope with a 20× objective. For confocal laser scanning microscopy, root meristems were mounted in 10 μg/mL propidium iodide. The process was performed according to the method described by Tian et al. [44 (link)]. In addition, to determine the number of cells belonging to the root meristem, root meristematic cortex cells were counted in a file extending from the QC to the first elongated cell excluded [46 (link)]. We quantified root cortical cell length in the maturation zone which has root hairs using 20 to 50 cells from 15 to 20 roots for each genetic background with Image J. Image processing was performed with the LSM image-processing software (Zeiss, Jena, Germany). We determined statistical significance by Student’s t test or one-way ANOVA (Tukey’s multiple comparison tests).
8
Subcellular Localization of TaPIN1 in Arabidopsis
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35S::TaPIN1-6a-CDS-GFP was inserted into the pMDC83 vector and infected into
Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [33] . Staining of roots with FM4-64 was performed as previously described [34] [35] [36] [37] [38] [39] . Confocal microscopy was performed on the root of the positive Arabidopsis plants with a Leica TCS SP5Ⅱ
(Richmond, IL, USA), and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880
Airyscan (Zeiss, German) with a 40× objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software (Zeiss, German).
Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [33] . Staining of roots with FM4-64 was performed as previously described [34] [35] [36] [37] [38] [39] . Confocal microscopy was performed on the root of the positive Arabidopsis plants with a Leica TCS SP5Ⅱ
(Richmond, IL, USA), and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880
Airyscan (Zeiss, German) with a 40× objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software (Zeiss, German).
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