Embryos were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with PBS and 0.3% PVP containing 0.5% Triton X-100 at room temperature for 20 min, and blocked in PBS and 0.3% PVP containing 1.0% BSA at room temperature for 1 h. These embryos were incubated with primary antibody in blocking solution overnight at 4°C. After washing 3 times with PBS and 0.3% PVP, the embryos were incubated with goat anti-rabbit IgG secondary antibody at room temperature for 2 h. The embryos were washed 3 times with PBS and 0.3% PVP, stained with 10 μg/ml Hoechst 33342 containing PBS and 0.3% PVP and examined under confocal microscope (Zeiss LSM 780 META). Images were processed by ZEN software. The antibodies used are listed in Supplementary Table S1 .
Lsm 780 meta
Manufactured by Zeiss
Sourced in Germany
The LSM 780 META is a confocal laser scanning microscope from Zeiss. It is designed for high-resolution imaging of fluorescent samples. The system features a range of laser excitation wavelengths and sensitive detectors for efficient signal collection.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm software, by Zeiss (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Alexa fluor 594 goat anti rat igg, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
Nci h460, by American Type Culture Collection (1 mentions)
Annexin 5 staining kit, by Beyotime (1 mentions)
Tcs sp8, by Leica (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
A3803, by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit igg, by Cell Signaling Technology (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Plan apochromat 10 0.45 m27, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
31 protocols using lsm 780 meta
1
Immunofluorescence Staining of Embryos
2
Immunofluorescent Staining of Oocyte Proteins
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Immunofluorescent staining was performed as described previously [39 (link), 40 (link)]. Oocytes were fixed in 4% paraformaldehyde in PBS buffer for 30 minutes at room temperature. After being permeabilized with 0.5% Triton X-100 for 20 minutes, they were then blocked in 1% BSA-supplemented PBS for 1 hour at room temperature. For staining of Spc24 or Mad2 or Bub3, oocytes were incubated overnight at 4°C with anti-Spc24 antibody (1:100); anti-Mad2 antibody (1:20); anti-Bub3 antibody (1:50), respectively. After three washes in washing buffer, oocytes were incubated with TRITC-conjugated goat anti-goat IgG (1:100) for 1 hours at room temperature. For α-tubulin staining, following incubation with anti-α-tubulin-FITC antibodies for 2 hours at room temperature, oocytes were washed 3 times in washing buffer, co-stained with Hoechst 33342 (10 mg/ml in PBS) for 15 min. These oocytes were mounted on glass slides and examined with a confocal laser-scanning microscope (Zeiss LSM 780 META, Germany).
3
Transient Expression of LrDFR1-GFP in Nicotiana benthamiana
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The pBinGFP4 vector with LrDFR1-GFP was transformed into the Agrobacterium tumefaciens strain EHA105, and transferred into Nicotiana benthamiana epidermal cells (Sheludko et al., 2007 (link)). Cultivation of the transformed N. benthamiana leaves was done for 2–6 days. For co-localization with membrane-localized marker, 35S: PIP2;1-mCherry construction was used (Huang et al., 2019 (link)). Assessment of transformed N. benthamiana epidermal cells was observed with confocal laser scanning microscopy (Zeiss LSM780 META, Jena, Germany). For staining of the nuclei, 10 mg/mL 4′6-diamidino-2-phenylindole (DAPI) was infiltrated into N. benthamiana leaves 6 h before observation.
Staining of proanthocyanidin was conducted as described by An et al. (2015) (link). Briefly, light-treated N. benthamiana leaves were decolorized in a solution of ethanol: glacial acetic acid (3:1). A dimethylaminocinnamaldehyde (DMACA) reagent staining solution (Sigma-Aldrich, St. Louis, MO, United States) was then added for staining.
Staining of proanthocyanidin was conducted as described by An et al. (2015) (link). Briefly, light-treated N. benthamiana leaves were decolorized in a solution of ethanol: glacial acetic acid (3:1). A dimethylaminocinnamaldehyde (DMACA) reagent staining solution (Sigma-Aldrich, St. Louis, MO, United States) was then added for staining.
4
Cytoskeletal Organization of MG63 Cells on HANF and SrHANF Matrices
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MG63 cells cultured on the HANF and SrHANF matrices were stained with rhodamine-phalloidin to observe the cytoskeletal organization. First, the matrices were fixed with 3.7 wt% paraformaldehyde in phosphate-buffered saline (PBS, 0.02 M pH = 7.4). Subsequently, the matrices were rinsed with 0.1 wt% Triton X-100 in PBS for 5 min and incubated with 1 wt% bovine serum albumin in PBS for 1 h to block nonspecific background staining. After blocking, the matrices were incubated with Alexa Fluor®488 phalloidin to stain the cytoskeleton through the binding of phalloidin to F-actin. Next, the matrices were incubated with 0.1 wt% 2-(4-amidinophenyl)-6-indolecarbamidine (DAPI) in PBS to stain the cell nuclei. Finally, the matrices were rinsed with PBS and observed by laser scanning confocal microscopy (LSCM, Zeiss LSM 780 META, Jena, Germany). The cytoskeleton and nuclei were stained green and blue, respectively.
5
Visualizing Immune Responses in Lymph Nodes
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QS-21-BODIPY (formulated in AS01) was produced as reported in ref. 6 (link). Frozen cryostat sections of draining lymph nodes (DLN) (5 mm) from mice immunised with QS-21- BODIPY were fixed in acetone. Ab staining of DLN sections (CD11b, CD169 – clone MOMA-1- and B cells, biotinylated rat anti-mouse CD45R/B220; BD Biosciences) was performed in PBS/2% donkey serum, overnight at 4 °C, in conjunction with Dylight594-conjugated streptavidin (for CD45R/ B220). Tissue sections were examined with a confocal laser scanning microscope (Zeiss LSM 780 Meta). Minor brightness and contrast adjustments were made using Zeiss and/or other routine image-manipulation software and were applied uniformly to the whole image.
6
Subcellular Localization of GmNHX6 Protein
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For subcellular localization of GmNHX6 protein, the open reading frame (ORF) of GmNHX6 was amplified by PCR using specific primers (Supplementary Table 2 ). The PCR product was then introduced into the pAN580 vector, which contains a green florescence protein (GFP) reporter gene, under the drive of CaMV 35S promoter for N-terminal GFP fusion (Zhang et al., 2016 (link)), using the ClonExpress II One Step Cloning Kit (Vazyme, China) according to the manufacturer’s protocol. To further determine the specific subcellular localization of GmNHX6, the fluorescent marker protein, Man1-mCherry, which is characteristic for the cis-Golgi (Tse et al., 2004 (link)), was co-expressed with GmNHX6. The transient expression of above proteins in Arabidopsis mesophyll protoplasts was performed following the published methods (Wu et al., 2009 (link)). Confocal imaging analysis was performed using a laser scanning microscope (Zeiss LSM780 META, Jena, Germany).
7
Immunofluorescence Analysis of Oocyte Proteins
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Oocytes were fixed in 4% paraformaldehyde dissolved in PBS (pH 7.4) for 30 min and then permeabilized in 0.5% Triton X-100 for 20 min. After 1 h in blocking buffer (PBS containing 1% BSA), the oocytes were incubated overnight at 4°C with primary antibodies (1:50-1:100 dilution), followed by incubation with Alexa Fluor 488-conjugated or Alex Fluor 594-conjugated secondary antibodies (1:200, Sigma-Aldrich) for 1–2 h at room temperature. Hoechst 33342 (10 mg/mL in PBS) was used for DNA counterstaining. For mitochondrial staining, oocytes were incubated for 30 min at 37°C in M2 medium supplemented with 200 nM MitoTracker Red (Invitrogen, Carlsbad, CA, USA). Next, the oocytes were mounted on glass slides and examined under a laser scanning confocal microscope (Zeiss LSM 780 META, Oberkochen, Germany). At least 20 oocytes were examined in each group, unless stated otherwise.
For measurement of immunofluorescent intensity, the signals from both control and experimental oocytes were acquired by performing the same immunostaining procedure and setting up the same parameters of confocal microscope. Data were analyzed by Image J software.
For measurement of immunofluorescent intensity, the signals from both control and experimental oocytes were acquired by performing the same immunostaining procedure and setting up the same parameters of confocal microscope. Data were analyzed by Image J software.
8
Multicolor Imaging of Arabidopsis Meristem
9
Subcellular Localization of LaOMT1 Protein
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For subcellular localization of LaOMT1 protein, the coding regions of LaOMT1 was amplified by PCR using specific primers (Table S1 ). The PCR product was then introduced into pAN580 vector carrying a double CaMV 35S promoter for N-terminal green fluorescent protein (GFP) fusion. The well-established fluorescent protein marker mCherry-HDEL for the ER was used [43 (link)]. Other fluorescent protein markers used for the cytoplasm (AtGAPC1-mcherry, At3g04120), the endosome (AtAra6-mcherry, At3g54840), and the peroxisome (AtPEX7-mcherry, At1g29260) were also constructed in the fusion with mCherry protein of a modified vector P16ΔS:sXVE:mCherryC. The transient expression of LaOMT1-GFP fusion genes in Arabidopsis mesophyll protoplasts was performed following previous method [72 (link)]. In addition, the LaOMT1-GFP construct was introduced into Agrobacterium tumefaciens strain EHA105, which was then transformed into epidermal cells of Nicotiana benthamiana [73 (link)]. The transformed protoplasts and N. benthamiana epidermal cells were observed with a confocal laser-scanning microscope (Zeiss LSM780 META, Jena, Germany).
10
Confocal Microscopy of Stained Samples
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Stained samples were visualized with a laser scanning confocal microscope (LSM 780 META Zeiss, Jena, Germany) with Zeiss Plan-Neofluar 10 × /0.3 and 20 × /0.5l dry lens objective. A 633-nm line of the HeNe laser was used to excite the dye (Cy5) and a laser 543-nmline to excite the Micro-Ruby. The images were obtained with a resolution of 1,024 × 1,024 pixels in the xy-plane and an interslice distance of 0.6–1 μm.
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