Transgenic Arabidopsis seedlings of 35 S:EIN3-GFP and 35 S:RAP2.12-GFP and were fixed in 4% PFA (pH 6.9) right after treatments, kept under gentle agitation for 1 h, were subsequently washed twice for 1 min in 1x PBS and stored in ClearSee clearing solution (xylitol 10% (w/v), sodium deoxycholate 15% (w/v) and urea 25% (w/v)44 (link). Seedlings were transferred to 0.01% Calcofluor White (in ClearSee solution) 24 h before imaging. Fluorescence was visualized using a Zeiss Observer Z1 LSM700 confocal microscope (oil immersion, ×40 objective Plan-Neofluar N.A. 1.30) with excitation at 488 nm and emission at 490–555 nm for GFP and excitation at 405 nm and emission at 400–490 nm for Calcofluor White. Within experiments, laser power, pinhole, digital gain and detector offset were identical for all samples. Mean GFP fluorescence pixel intensity in root tips was determined in similar areas of ~17,000 μm2 between epidermis layers using ICY software (http://icy.bioimageanalysis.org/ ).
Observer z1 lsm700 confocal microscope
Manufactured by Zeiss
The Observer Z1 LSM700 is a confocal microscope from Zeiss. It is designed for high-resolution imaging of samples. The microscope utilizes laser scanning technology to capture detailed images of specimens.
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Lab products found in correlation
Peroxidase from horseradish, by Merck Group (1 mentions)
2 ethanesulfonic acid mes, by Merck Group (1 mentions)
Bx50wi, by Olympus (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Daf fm diacetate, by Bio-Connect (1 mentions)
Acetylated tubulin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Huc d, by Merck Group (1 mentions)
Zen black, by Zeiss (1 mentions)
4 protocols using observer z1 lsm700 confocal microscope
1
Visualizing EIN3 and RAP2.12 Regulation in Arabidopsis
2
Visualizing Subcellular Hydrogen Peroxide
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DAB staining: DAB is oxidized by H2O2 in the presence of peroxidases to generate a dark brown precipitate (Thordal-Christensen et al., 1997 ). Seedlings were incubated with 1 mg·mL−1 DAB (Sigma-Aldrich) in 20-mM 2-ethanesulfonic acid (MES; Sigma-Aldrich) buffer (pH 6.2) supplemented by 10 units·mL−1 peroxidase from horseradish (Sigma-Aldrich) for 1 h. Seedlings were then rinsed with MES buffer for 1 min twice before imaging (Dubreuil-Maurizi et al., 2011 (link)). Olympus BX50WI was used to visualize DAB staining and images were taken under 10× objective lens.
Confocal imaging of mt-roGFP2-Orp1: to visualize subcellular levels of H2O2, confocal imaging of 5-day-old mt-roGFP2-Orp1 Arabidopsis seedlings was performed right after experimental time-points with a Zeiss Observer Z1 LSM700 confocal microscope (oil immersion, 40× objective Plan-Neofluar N.A. 1.30). RoGFP2-Orp1 was excited sequentially at 405 and 488 nm and emission was recorded at 505–535 nm. The 405/488 ratio within root tips was determined in similar areas of ∼5,000 μm2 using ICY software (http://icy.bioimageanalysis.org ).
Confocal imaging of mt-roGFP2-Orp1: to visualize subcellular levels of H2O2, confocal imaging of 5-day-old mt-roGFP2-Orp1 Arabidopsis seedlings was performed right after experimental time-points with a Zeiss Observer Z1 LSM700 confocal microscope (oil immersion, 40× objective Plan-Neofluar N.A. 1.30). RoGFP2-Orp1 was excited sequentially at 405 and 488 nm and emission was recorded at 505–535 nm. The 405/488 ratio within root tips was determined in similar areas of ∼5,000 μm2 using ICY software (
3
Quantifying Intracellular Nitric Oxide Levels
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Intracellular NO levels were visualized using DAF-FM diacetate (7'-difluorofluorescein diacetate, Bio-Connect). Seedlings were incubated in the dark for 15 min under gentle agitation in 10 mM Tris-HCl buffer (pH 7.4) containing 50 μM DAF-FM DA and subsequently washed twice for 5 min 10 mM Tris-HCl buffer (pH 7.4). Several roots of all treatments/genotypes were mounted in 10 mM Tris-HCl buffer (pH 7.4) on the same microscope slide. Fluorescence was visualized using a Zeiss Observer Z1 LSM700 confocal microscope (oil immersion, 40x objective Plan-Neofluar N.A. 1.30) with excitation at 488 nm and emission at 490–555 nm. Roots incubated and mounted in 10 mM Tris-HCl buffer (pH 7.4) without DAF-FM DA were used to set background values where no fluorescence was detected. Within experiments, laser power, pinhole, digital gain and detector offset were identical for all samples. Mean DAF-FM DA fluorescence pixel intensity in root tips was determined in similar areas of ~17,000 μm2 between epidermis layers using ICY software (http://icy.bioimageanalysis.org/ ).
4
Whole-mount Immunostaining of Embryos
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For whole-mount immunostaining, embryos were fixed in 4% PFA at 4°C overnight. Embryos were placed in blocking solution (PBS containing 0.5% Triton X-100, 1% DMSO, 5% goat serum, 1% bovine serum albumin, and 4% sodium azide) for 2 hours at room temperature. Primary antibodies against HuC/D (Sigma, 1:1000), acetylated tubulin (Sigma, 1:500), Col2α1 (Developmental Studies Hybridoma Bank 1:100), Fibronectin (Sigma, 1:400) were diluted in blocking solutions and incubated 1 to 2 days. Alexa Fluor 488-conjugated and Alexa Fluor 555-conjugated anti-mouse or anti-rabbit secondary antibodies (Molecular Probes, Grand Island, NY, USA) were used at 1:500 dilution. Nuclear staining was performed using TO-PRO-3 iodide at a 1:1000 dilution incubated for 1 hour at room temperature. Images were acquired using a Zeiss Observer Z1 LSM 700 confocal microscope (40X 1.1 NA W or 20X 0.8 NA objectives). Regions of interest in stained embryos/larvae were imaged with several confocal optical z-sections. Zen Black (Zeiss) software was used to produce maximum intensity image projections of the z-section volumes.
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