KGN and SVOG cells were fixed by treatment with paraformaldehyde at a concentration of 4% at 37°C for 20 min. Permeabilization and blocking were accomplished after treatment with 0.3% Triton X-100 and 10% BSA at room temperature for 30 min. The samples were incubated at 4°C overnight with specific primary antibodies. Subsequently, the antibodies were labeled at room temperature through treatment with Alexa Fluor secondary antibody for 1 h. Nuclei were stained with DAPI. A laser confocal microscope (ANDOR, UK) was utilized to capture images. Table S4 shows the antibodies used for immunofluorescence.
Laser confocal microscope
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Laser Confocal Microscope is an advanced optical imaging instrument that utilizes a focused laser beam to scan and capture high-resolution, three-dimensional images of microscopic samples. It functions by illuminating and detecting light from a specified focal plane within the sample, allowing for the acquisition of optical sections with minimal interference from out-of-focus regions.
Automatically generated - may contain errors
8 protocols using laser confocal microscope
1
Immunofluorescence Staining of KGN and SVOG Cells
2
Immunofluorescence Staining of KGN Cells
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The processed KGN cells were washed by PBS and fixed for 20 min in 4% paraformaldehyde at room temperature. After that, cells were permeabilized in 0.3% Triton X-100 and blocked by 5% bovine serum albumin in PBS for 1 h. Then samples were incubated with specific primary antibody at 4°C overnight and Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Nuclei were counterstained with DAPI. Images were visualized under a laser confocal microscope (ANDOR, UK). The details of antibodies are listed in Supplementary Table S3 .
3
Subcellular Localization of TaWRKY31
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For the subcellular localization analysis of TaWRKY31, we designed primers with appropriate double restriction enzyme sites, XbaI and KpnI, based on the full-length TaWRKY31 and p35s-1301-GFP vector. The target gene sequence was amplified using these primers. The complete TaWRKY31 ORF without the termination codon was then inserted into the p35s-1301-GFP vector using the ClonExpressII One Step Cloning Kits (Vazyme, Nanjing, China). The p35s-1301-TaWRKY31-GFP plasmid and p35s-1301-GFP empty vector were transformed into Agrobacterium GV3101 and subsequently infiltrated into the abaxial surface of Nicotiana benthamiana leaves using 1-mL needleless syringes along with NLS-mCherry and P19 as controls [53 (link)]. The transformed tobacco plants were initially grown in a dark environment at 22℃ for 24 h and then transferred to controlled conditions with a temperature of 22℃, a photoperiod of 16 h/8 h, and an illumination intensity of 180 µmol·m− 2·s− 1 for 2 d. Green fluorescent protein (GFP) signals were observed using a laser confocal microscope (Andor, Belfast, UK) at an excitation wavelength of 488 nm. The specific primers used for this analysis are listed in Supplementary Table S1 .
4
Cloning and expression of TaWRKY133 in N. benthamiana
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The CDS of TaWRKY133 without the stop codon was cloned from the wheat variety Pubing 143 by PCR using specific primers containing the XbaI and KpnI restriction sites and homologous arms listed in the Supplementary Table . Then, the cloned sequence was ligated with the p35S-1301-GFP vector using homologous recombination to obtain a recombinant plasmid. After sequencing, the recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 through the heat shock method. Agrobacterium GV3101 containing the pYJmCherry vector connected with the NLS was kindly provided by Professor Jiang Yuanqing. Two kinds of Agrobacterium were mixed in equal volumes, resuspended in infiltration buffer (0.15 mM acetosyringone, 10 mM MgCl2 and 10 mM MES-KOH) and then infiltrated into leaves of 28-day-old N. benthamiana plants with a needleless syringe. GFP and mCherry signals were observed under a laser confocal microscope (Andor, Belfast, UK).
5
Fluorescent DDGC Probe Detection
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DDGC probes were synthesized by RiboBio and labeled with Cy3. Then, assays were conducted using a fluorescence in situ hybridization (FISH) kit (RiboBio) following the manufacturer's protocols. The samples were observed using a laser confocal microscope (ANDOR, UK).
6
Fluorescent in situ Hybridization of HCP5
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A mix of probes targeting HCP5 was synthesized and labeled with Cy3 (RiboBio, China). The experiment was performed using Fluorescent in situ Hybridization Kit (RiboBio, China) according to the manufacturer's instructions and visualized under a laser confocal microscope (ANDOR, UK). The Cy3-labeled U6 and 18S probes were hybridized simultaneously as controls.
7
Localization of TaWRKY46 Protein in Wheat Leaf Protoplasts
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The cDNA encoding full-length TaWRKY46 without the stop codon was inserted into the pTF486-GFP vector [54 (link)] after it was cloned by PCR using specific primers (Supplementary Table S2 ) with BamHI restriction site. Wheat leaf protoplasts were isolated from 10-day-old seedlings according to the method of Shan [55 (link)]. pTF486-TaWRKY46-GFP was transformed into protoplasts by the PEG-mediated method and then incubated at 22 °C for 12 h in the dark. Empty vector was used as a control. GFP fluorescence signals were visualized using a laser confocal microscope (Andor, Belfast, UK) at an excitation wavelength of 488 nm.
8
Detecting circSTK40 in THESCs
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THESCs were hybridized overnight with a mixture of Cy3-labeled circSTK40, U6, or 18S probes (RiboBio, China). All operations were completed using a Fluorescence in situ Hybridization Kit (RiboBio, China) according to the manufacturer’s instructions. Specimens were visualized using a laser confocal microscope (Andor, UK).
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