NTHi strains were grown on chocolate agar for 16 h, in the absence or presence of thymidine, and a colony was aseptically spread with an inoculation loop over a drop of distilled water on a microscopy slide. Samples were air-dried and stained for 15 min in the darkness with the cell-permeable fluorescent nucleic acid stain SYTO 9 (Life Technologies), following the manufacturer's instructions. Samples were washed twice with distilled water and fluorescence was observed by confocal laser microscopy. Images were acquired using a Leica TCS-SL filter-free spectral confocal laser-scanning microscope (Leica Microsystems) equipped with a 488 nm argon laser, 543 nm and 633 nm He/Ne lasers (Centres Científics i Tecnològics-Campus de Bellvitge, Universitat de Barcelona, Spain) using a 63× magnification oil immersion objective (1.4 numerical aperture), and an image resolution of 1024 × 1024 pixels. Images were acquired randomly and analyzed using the Leica Confocal Software 2.5 (Leica Microsystems).
Confocal software 2
Manufactured by Leica
Leica Confocal Software 2.5 is a software package designed to operate Leica confocal microscopes. It provides the core functionality required to control the microscope hardware, acquire and process confocal images.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop, by Zeiss (3 mentions)
Tcs sl filter free spectral confocal laser scanning microscope, by Leica (2 mentions)
He ne lasers, by Leica (2 mentions)
Mzfliii, by Leica (2 mentions)
Analysis doku 3, by Olympus (2 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Tcs spii, by Leica (1 mentions)
Rm2135 rotary microtome, by Leica (1 mentions)
Mzfliii stereomicroscope, by Leica (1 mentions)
5 protocols using confocal software 2
1
Fluorescence Microscopy of NTHi Cells
2
Confocal microscopy of bacterial biofilms
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Overnight bacterial cultures were used to inoculate an eight-well chambered cover glass for confocal image analysis (Ibidi GmbH) to a final OD620 of 0.01 in 2 ml of sBHI. The plates were incubated at 37°C for 24 h without shaking. After incubation, the wells were washed with distilled water and stained for 15 min in the dark with the cell-permeating fluorescent dye SYTO 17 Red Fluorescent Nucleic Acid Stain in accordance with the manufacturer’s instructions (Molecular Probes).
Samples were washed three times to remove nonspecific staining, and fluorescence was observed by confocal laser microscopy. Images of the double-labeled sections were acquired with a Leica TCS-SL filter-free spectral confocal laser scanning microscope (Leica Microsystems, Inc.) with a 488-nm argon laser, 543- and 633-nm He/Ne lasers (Centres Científics i Tecnològics, Campus de Bellvitge, Universitat de Barcelona, Barcelona, Spain), a 63× oil immersion objective (1.4 numerical aperture), and an image resolution of 1,024 by 1,024 pixels. The images were acquired randomly from the cover glass surface and analyzed with the Leica Confocal Software 2.5 (Leica Microsystems, Inc.).
Samples were washed three times to remove nonspecific staining, and fluorescence was observed by confocal laser microscopy. Images of the double-labeled sections were acquired with a Leica TCS-SL filter-free spectral confocal laser scanning microscope (Leica Microsystems, Inc.) with a 488-nm argon laser, 543- and 633-nm He/Ne lasers (Centres Científics i Tecnològics, Campus de Bellvitge, Universitat de Barcelona, Barcelona, Spain), a 63× oil immersion objective (1.4 numerical aperture), and an image resolution of 1,024 by 1,024 pixels. The images were acquired randomly from the cover glass surface and analyzed with the Leica Confocal Software 2.5 (Leica Microsystems, Inc.).
3
Microscopic Imaging of GUS, GFP, and Esculin in Plants
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Pictures of GUS plants were taken with a Leica MZFLIII stereomicroscope (Leica Microsystems) or with a Zeiss Axioskop (Carl Zeiss Jena GmbH). Pollen tubes for length measurements were analyzed by light microscopy (Zeiss Axioskop; Carl Zeiss Jena). Pollen stained with iodine potassium iodide solution were analyzed in bright field at 1000x magnification with a Zeiss Axioskop (Carl Zeiss Jena GmbH). Images of GFP-reporter plants and GFP expressing pollen tubes were taken on a Leica 765 TCS SPII confocal laser scanning microscope (Leica Microsystems) and processed with Leica Confocal Software 2.5. A 488-nm argon laser was used for excitation of GFP and chlorophyll autofluorescence. The 415-nm diode was used for the excitation of esculin fluorescence. Detection windows ranged from 497 to 526 nm for GFP, from 682 to 730 nm for chlorophyll autofluorescence and from 424 to 469 nm for esculin. Images of GFP and esculin fluorescence for the pollen tube esculin uptake assay were taken in a sequential mode. Image processing was done using analySIS Doku 3.2 software (Soft Imaging System, Münster) and GIMP2.10 (https://www.gimp.org/ ).
4
Microscopic Analysis of GUS Plants
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GUS plants were analysed under a stereomicroscope (Leica MZFLIII; Leica Microsystems) or a microscope (Zeiss Axioskop; Carl Zeiss Jena GmbH). Images were processed using the analySIS Doku 3.2 software (Soft Imaging System, Münster, Germany).
Images of protoplasts and GFP-reporter plants were taken on a confocal laser scanning microscope (Leica TCS SPII; Leica Microsystems) using a 488nm argon laser for excitation and processed with Leica Confocal Software 2.5. Detection windows ranged from 497 to 526nm for GFP and from 682 to 730nm for chlorophyll autofluorescence.
Images of protoplasts and GFP-reporter plants were taken on a confocal laser scanning microscope (Leica TCS SPII; Leica Microsystems) using a 488nm argon laser for excitation and processed with Leica Confocal Software 2.5. Detection windows ranged from 497 to 526nm for GFP and from 682 to 730nm for chlorophyll autofluorescence.
5
Confocal Microscopy of Fluorescent Biological Samples
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Images of GFP-reporter plants, GFP expressing yeast strains and protoplasts were taken on a Leica 765 TCS SPII confocal laser scanning microscope (Leica Microsystems) and processed with Leica Confocal Software 2.5. A 488-nm argon laser was used for excitation of GFP, chlorophyll autofluorescence and propidium iodide. The 415-nm diode was used for the excitation of esculin fluorescence. Detection windows ranged from 497 to 526 nm for GFP, from 682 to 730 nm for chlorophyll autofluorescence, from 589 to 684 nm for propidium iodide, and from 424 to 469 nm for esculin. Images of GFP and esculin fluorescence for the esculin uptake assay were taken in a sequential mode. Fluorescence intensities of GFP and esculin were quantified in three regions of a defined size in each protoplast image using ImageJ 1.50b (Schneider C.A. et al., 2012 (link)). Images of GUS plants were taken with a Zeiss Axioskop (Carl Zeiss Jena GmbH) or with a Leica MZFLIII stereomicroscope (Leica Microsystems). For cross-sections stained roots were embedded into Technovit as described (Beeckman and Viane, 2000 (link); Ühlken et al., 2014 (link)) and sections were cut using a Leica RM2135 rotary microtome. Image processing was done using the analySIS Doku 3.2 software (Soft Imaging System, Münster), ImageJ 1.50b (Schneider C.A. et al., 2012 (link)) and GIMP2.82 .
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