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).
Two photon laser confocal microscope
Manufactured by Zeiss
Sourced in Germany
The two-photon laser confocal microscope is a high-resolution imaging system designed for advanced biological and materials research. It utilizes a focused laser beam to excite fluorescent molecules within a sample, enabling three-dimensional imaging with improved optical sectioning and reduced photodamage compared to traditional confocal microscopy. The core function of this instrument is to provide researchers with a powerful tool for visualizing and analyzing complex structures and dynamic processes at the cellular and subcellular levels.
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5 protocols using two photon laser confocal microscope
1
Subcellular Localization of MdTLP7 Protein
2
Subcellular Localization of MdWRKY31 Protein
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To explore the subcellular localization of MdWRKY31, the open reading frame (ORF) of MdWRKY31 was amplified from ‘Royal Gala’ apple tissue culture seedlings using PCR in conjunction with MdWRKY31-F and MdWRKY31-R as primers (Table S1 ). Based on MdWRKY31 sequences, the Gateway system was used to insert the MdWRKY31 ORF into the pENTRTM Directional TOPO vector with MdWRKY31-TOPO upstream and downstream primers. The recombination reaction (LR) was used to insert the MdWRKY31 gene into the PAL1107 vector to construct a MdWRKY31-RFP vector for subcellular localization.
Bacterial solutions of MdWRKY31-RFP and P19 were cultured for 12 h. After centrifugation, the Agrobacterium liquid was suspended in an MMA liquid medium and mixed in a 1:1 proportion of MdWRKY31-RFP:P19 for 4–5 h. A 500 μl suspension liquid was injected into the leaves of Nicotiana benthamiana. Images were taken with a two-photon laser confocal microscope (Carl Zeiss;German) at 3–4 days after the plants were transferred to a growth chamber with a temperature of 25 °C and a 16-h photoperiod.
Bacterial solutions of MdWRKY31-RFP and P19 were cultured for 12 h. After centrifugation, the Agrobacterium liquid was suspended in an MMA liquid medium and mixed in a 1:1 proportion of MdWRKY31-RFP:P19 for 4–5 h. A 500 μl suspension liquid was injected into the leaves of Nicotiana benthamiana. Images were taken with a two-photon laser confocal microscope (Carl Zeiss;German) at 3–4 days after the plants were transferred to a growth chamber with a temperature of 25 °C and a 16-h photoperiod.
3
Adipocyte Visualization and Quantification
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On d 8 post-induction, adipocytes grown on slides were washed twice with D-Hank’s and subsequently fixed with 4% paraformaldehyde for 1 h at RT. Adipocytes were stained with bodipy (1 µg/mL) for 30 min and then with DAPI (1 µg/mL) for 5 min. The cultures were photographed with a two-photon laser confocal microscope (Zeiss, Germany).
The differentiated adipocytes were stained with oil red O on d 8 post-treatment. Cells were washed twice with D-Hank’s and subsequently fixed with 4% paraformaldehyde for 1 h at RT. Following fixation, the cells were washed twice with D-Hank’s and subsequently stained with 0.6% oil red O solution for 1 h. Haematoxylin staining was performed to visualize the cell nuclei. After washing, the cultures were photographed with an inverted microscope (Olympus, Japan). The stained oil red O was quantified and expressed as mmol/g total protein.
The differentiated adipocytes were stained with oil red O on d 8 post-treatment. Cells were washed twice with D-Hank’s and subsequently fixed with 4% paraformaldehyde for 1 h at RT. Following fixation, the cells were washed twice with D-Hank’s and subsequently stained with 0.6% oil red O solution for 1 h. Haematoxylin staining was performed to visualize the cell nuclei. After washing, the cultures were photographed with an inverted microscope (Olympus, Japan). The stained oil red O was quantified and expressed as mmol/g total protein.
4
Measuring Preadipocyte Proliferation and Viability
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EdU, which is a rather new thymidine analog that can be incorporated into DNA during active DNA synthesis, has widely been used to trace the cell proliferation ability [21 (link)]. In this study, subconfluent preadipocytes were treated with different SB doses for 24 h. Then, the cells were incubated with 10 µM EdU for 2 h. Cell increment could be detected via a chemical reaction with Andy FluorTM 488 Azide (green fluorescence). The nuclei were stained with Hoechst 33,342 (blue fluorescence). Pictures were photographed with a two-photon laser confocal microscope (Zeiss, Germany).
CCK-8 analysis was conducted to examine the cell viability. After treating subconfluent preadipocytes for 24 h with serial concentrations of SB, CCK-8 solution (Vazyme, Nanjing) was added to the cells. Following incubation with CCK-8 for 3 h, the absorbance at 450 nm was measured with a microplate reader (BioTek, USA).
CCK-8 analysis was conducted to examine the cell viability. After treating subconfluent preadipocytes for 24 h with serial concentrations of SB, CCK-8 solution (Vazyme, Nanjing) was added to the cells. Following incubation with CCK-8 for 3 h, the absorbance at 450 nm was measured with a microplate reader (BioTek, USA).
5
Subcellular Localization of Maize PIF4 and PIF5
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Plasmids 35Spro::ZmPIF4-GFP and 35Spro:ZmPIF5-GFP were generated by PCR-amplifying the coding region of ZmPIF4 and ZmPIF5 from 35Spro::ZmPIF4-3FLAG or 35Spro:ZmPIF5-3FLAG using primer pairs GFP-ZmPIF4-F and GFP-ZmPIF4-R, and GFP-ZmPIF5-F and GFP-ZmPIF5-R. The fragments containing the coding region of ZmPIF4 and ZmPIF5 were then inserted into pPZP211-35Spro::GFP digested with BamHI and XbaI. The various plasmids were then transformed into the Agrobacterium strain GV3101 and infiltrated into leaves of N. benthamiana together with the 35Spro::H2B-Cherry (Howe et al., 2012 (link)). Three days after infiltration, the N. benthamiana plants were transferred into dark conditions for 12 h, the lower epidermis of the infiltrated leaf was peeled, and the fluorescence signals were observed with a two-photon laser confocal microscope (Zeiss).
For subcellular localization analysis of ZmPIF4 and ZmPIF5 in protoplasts, 30-day-old Arabidopsis wild-type plants (Col-0) grown under short day (8-h light/16-h dark) conditions were used to isolate the protoplasts. Plasmids 35Spro::ZmPIF4-GFP and 35Spro:ZmPIF5-GFP were transformed into the protoplasts using PEG method (Yoo et al., 2007 (link)). Incubated for 18 h in darkness to allow the constructs to be expressed, and then the fluorescent signals were observed with a laser confocal microscope (Zeiss).
For subcellular localization analysis of ZmPIF4 and ZmPIF5 in protoplasts, 30-day-old Arabidopsis wild-type plants (Col-0) grown under short day (8-h light/16-h dark) conditions were used to isolate the protoplasts. Plasmids 35Spro::ZmPIF4-GFP and 35Spro:ZmPIF5-GFP were transformed into the protoplasts using PEG method (Yoo et al., 2007 (link)). Incubated for 18 h in darkness to allow the constructs to be expressed, and then the fluorescent signals were observed with a laser confocal microscope (Zeiss).
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