BiFC constructs were expressed in mesophyll protoplasts as described above. The protoplasts were incubated with MitoTracker orange CMTMRos (Thermo Fisher Scientific) for mitochondrial staining at 37 °C for 10 min followed by 26 °C for 20 min. Confocal images were taken using a DM6000B/SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). BiFC and MitoTracker fluorescence were imaged with a 488 and 555 nm laser excitation, and emission fluorescence was captured by 500–520 and 560–580 nm band-pass emission filters, respectively.
Dm6000b sp5 confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany
The DM6000B/SP5 confocal laser scanning microscope is a sophisticated imaging instrument designed for high-resolution microscopy. It utilizes laser excitation and advanced detection systems to capture detailed images of samples at the cellular and subcellular level. The core function of this microscope is to provide researchers and scientists with a powerful tool for studying the structure and dynamics of a wide range of biological specimens.
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6 protocols using dm6000b sp5 confocal laser scanning microscope
1
Visualizing Mitochondrial Localization of BiFC Constructs
2
BiFC Protein Interaction Visualization
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Young leaves of four-weeks-old N. benthamiana plants were infiltrated with A. tumefaciens, transformed with BiFC vectors grown on selective medium, and protein expression visualized with DM6000B/SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Laser excitation was 490–515 nm for YFP and 555 nm for RFP; emission fluorescence was captured by 500 to 520 nm and 555 to 580 nm band-pass emission filters, respectively.
3
Confocal Imaging of GFP Transgenic Lines
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35S:GFP, 35S:NDPK1-GFP, 35S:NDPK2-GFP, and 35S:NDPK3-GFP transgenic cell culture lines were analysed with a DM6000B/SP5 confocal laser scanning microscope (Leica Microsystems).
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35S:GFP, 35S:NDPK1-GFP, 35S:NDPK2-GFP, and 35S:NDPK3-GFP transgenic cell culture lines were analysed with a DM6000B/SP5 confocal laser scanning microscope (Leica Microsystems).
4
Confocal Imaging of Fluorescent Proteins
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All confocal images were taken using a DM6000B/SP5 confocal laser-scanning microscope, and 63× water objective was used (Leica Microsystems, Wetzlar, Germany). For images depicted in Fig. 6 , 514 nm laser at 10% and 569 nm laser at 10% were used for YFP, and RFP signals, respectively. Emission ranges were collected between 519 and 558 nm with a gain of 697 V for YFP signals and between 568 and 625 nm with a gain of 637 V for RFP signals. For images depicted in Fig. 7 , 514 nm laser at 40% and 561 nm laser at 20% were used for YFP, and RFP signals, respectively. Emission ranges were collected between 520 and 565 nm with a gain of 643 V for YFP signals and between 575 and 640 nm with a gain of 755 V for RFP signals.
5
SlJUB1 Expression in N. benthamiana
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The SlJUB1 coding sequence (without stop codon) was amplified by PCR from S. lycopersicum cv. Moneymaker leaf cDNA (primers listed in Table S1 ) and cloned into pDONR 207 using BP clonase (Invitrogen). The sequence‐confirmed entry vector was recombined into pK7FWG2 (Karimi et al., 2002 ) using LR reaction mix II (Life Technologies). The recombined plasmid (35S:SlJUB1‐GFP) was transformed into Agrobacterium tumefaciens (strain GV3101) and then used for infiltration of N. benthamiana leaves (Senthil‐Kumar and Mysore, 2014 ). GFP signal was analysed using a Leica DM6000B/SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).
6
Visualizing Autophagy in Arabidopsis
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Seeds of Arabidopsis thaliana (Col-0) transgenic by PromUBQ10:GFP-ATG8a construct ( 21 (link)
) (carrying coding region of the reporter gene of green fluorescent protein (GFP) fused to ATG8A as a molecular marker of autophagy) was sown and grown as
described above for non-transgenic seeds. Plates containing 7-day old seedlings were subjected to thermopriming and then to the growth chamber for about 48 hours.
Seedlings were treated in an MS culture medium containing 1 µM conconamycin A to raise the pH in vacuolar lumens to inhibit vacuolar hydrolases.
This resulted in the accumulation of autophagic bodies in the vacuoles during imaging. Pictures were taken 48 h after thermopriming by Leica DM6000B/SP5 confocal
laser scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany). SAM of three seedlings was screened for each primed and unprimed (control) state.
) (carrying coding region of the reporter gene of green fluorescent protein (GFP) fused to ATG8A as a molecular marker of autophagy) was sown and grown as
described above for non-transgenic seeds. Plates containing 7-day old seedlings were subjected to thermopriming and then to the growth chamber for about 48 hours.
Seedlings were treated in an MS culture medium containing 1 µM conconamycin A to raise the pH in vacuolar lumens to inhibit vacuolar hydrolases.
This resulted in the accumulation of autophagic bodies in the vacuoles during imaging. Pictures were taken 48 h after thermopriming by Leica DM6000B/SP5 confocal
laser scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany). SAM of three seedlings was screened for each primed and unprimed (control) state.
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