The MtPHO1.1 and MtPHO1.2 genes were amplified from genomic DNA using gene-specific primers (Supplemental Table S3 ), cloned into the pENTR2B (Invitrogen) by In-Fusion polymerase (Takara Bio), and inserted into pMDC83 plasmid by Gateway cloning (InVitrogen). For transient expression, N. benthamiana plants were infiltrated with Agrobacterium tumefaciens as previously described (Arpat et al., 2012 (link)). Pi and nitrate export assays were performed as previously described (Arpat et al., 2012 (link)). Pi concentration in solution was quantified by the molybdate assay (Ames, 1966 ), whereas nitrate was quantified by first converting nitrate to nitrite using commercial nitrate reductase from Aspergillus niger (Sigma) followed by nitrite quantification using sulfanilamide (Barthes et al., 1995 ). Confocal imaging was performed using a Zeiss LSM 700 instrument with Apochromat 63 × NA1.2 water immersion or multi-immersion objective.
Lsm 700 instrument
Manufactured by Zeiss
Sourced in Germany
The LSM 700 is a laser scanning microscope designed for high-resolution imaging of biological samples. It features a compact, modular design and offers a range of imaging modes, including confocal, multiphoton, and super-resolution.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated polyclonal goat anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions)
Gateway cloning, by Thermo Fisher Scientific (1 mentions)
Pentr2b, by Thermo Fisher Scientific (1 mentions)
Nitrate reductase from aspergillus niger, by Merck Group (1 mentions)
Facs lsrii sorp system, by BD (1 mentions)
Prism 8, by GraphPad (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
5 protocols using lsm 700 instrument
1
Functional Characterization of Phosphate Transporters
2
EGFR Expression Analysis via Flow Cytometry
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A total of 2 × 106 cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min at room temperature in the dark. The cells were washed and incubated with 1% BSA for 1 h at room temperature. After incubation, the cells were washed with PBS and incubated with 0.5 nM anti-EGFR (199.125; Thermo Scientific, Rockford, IL, USA) for 3 h at room temperature. The cells were then incubated with Alexa fluor 488-conjugated polyclonal goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., USA) for 1 h at room temperature. After washing with PBS, fluorescence was detected with BD FACS LSRII SORP system (Becton Dickinson Company, New Jersey, USA), and confocal microscopy was performed using an LSM 700 instrument (Carl Zeiss, Oberkochen, Germany).
3
Quantitative Fluorescence Imaging Analysis
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CLSM images were obtained using a Carl Zeiss LSM 700 instrument. The raw data were analyzed using Zeiss LSM ZEN 2009 software, GraphPad Prism 8.0 software, and the Image J program. To ensure consistency, all sample fluorescence intensities were measured and analyzed using the same CLSM parameters and Image J program. All the experiments were repeated more than three times.
4
Immunofluorescent Detection of EGFR
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Formalin-fixed, paraffin-embedded tissue samples were sectioned into serial 4-mm slices and placed on microscope slides. After deparaffinization in xylene and rehydration in alcohol, tissue sections underwent antigen retrieval for 30 min in Tris-EDTA buffer titrated to pH 9.0 at 97°C and cooled with tap water. Non-specific antigen reactions were blocked by a 1-h incubation with M.O.M. mouse IgG blocking buffer. The slides were then incubated with the anti-EGFR (MA5-13319; Thermo Scientific, Rockford, IL, USA) at a dilution of 1:500 for 12–16 h at 4°C. The slides were then incubated with Alexa fluor 488-conjugated polyclonal goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., USA) at a dilution of 1:100 for 1 h at room temperature. After washing with PBS, the slides were examined under confocal microscopy using LSM 700 instrument (Carl Zeiss, Oberkochen, Germany).
5
Imaging Adherence of Sulfurimonas sputigena
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For widefield microscopy and laser scanning confocal microscopy (LSCM) analyses of S. sputigena association, GECs were seeded at 1 × 10 5 on glass coverslips in 12-well plates and grown until ~60% confluent. Cells were infected with BacLight green-labeled S. sputigena at MOIs of 10, 50, and 100 for 1 h. Coverslips were washed 3 times with phosphate-buffered saline (PBS) and fixed for 10 min in 4% paraformaldehyde. Following a 20 min block in 5% bovine serum albumin, actin was labeled using 1:400 Alexa Fluor 568-phalloidin (Invitrogen) for 1 h at room temperature. After 4 washes in PBS, coverslips were mounted using ProLong gold with 4′,6diamidino-2-phenylindole (DAPI) mounting medium (Invitrogen). Images were acquired using either a Zeiss Axio Observer 7 or a Zeiss LSM700 instrument as part of the VCU Microscopy Core Facility.
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