HEK293T cells were seeded in 25-mm poly-L-lysine-treated coverslips and transfected at 70–80% confluency using Lipofectamine 2000 (ThermoFischer Scientific) with DNA for OLPVR1 fused to GFP at the C-terminal in pcDNA3.1, and pDsRed2-ER vector (Clontech, #632409) in a 6:1 ratio. After one day of expression, the cells were incubated for 30 min at 37 °C in standard bath solution with Hoescht 33342 (1/5000 dilution) and CellMask Deep Red (1/1000 dilution; #C10046 ThermoFischer Scientific). Hoescht 33342 was applied to stain the cell nucleus and CellMask Deep Red to stain the plasma membrane. pDsRed2-ER expression was used to label the ER. After the incubation, the cells were washed twice with standard bath solution and imaged immediately. Images were acquired using Zeiss Zen Black 2.3 software through the objective Plan-Apochromat 63x/1.40 Oil DIC M27 of a Zeiss LSM 710 Laser Scanning Confocal Microscope equipped with the lasers DPSS 561-10 nm, Argon 488 nm, Diode 405-30 nm and Helium Neon 633 nm, as well as two descanned GaAsp detectors ([570-610 nm], [500-550 nm]) and a descanned PMT detector set to 415-485 nm or 642-740 nm. Composite images result from channel merging with ImageJ software.
Pdsred2 er vector
Manufactured by Takara Bio
Sourced in United States
The PDsRed2-ER vector is a fluorescent protein expression vector designed for selective targeting and visualization of the endoplasmic reticulum (ER) in living cells. The vector expresses the DsRed2 fluorescent protein fused to an ER-targeting sequence, enabling the specific labeling and tracking of the ER compartment within the cellular environment.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Mcherry sec61β, by Addgene (1 mentions)
Memerald sec61β, by Addgene (1 mentions)
Dapi antifade solution, by Merck Group (1 mentions)
4 protocols using pdsred2 er vector
1
Visualizing ER-Membrane Protein Localization in HEK293T Cells
2
Subcellular Localization of Thyroperoxidase
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HEK293 cells overexpressing wild type or mutant TPO were seeded on a chambered coverglass and then transfected with pDsRed2-ER Vector (Clontech laboratories, Palo Alto, CA, 632409). Twenty-four hours after transfection, the cells were fixed with 10% formalin-phosphate buffer solution (PBS) for 15 minutes at room temperature. After washing three times with PBS, the cells were treated with or without 0.5% Triton-X100 for permeabilization, blocked with BlockingOne (Nacalai tesque, Kyoto, Japan, 03953-66), and incubated with anti-TPO antibody (Abcam, Cambridge, MA, USA; ab109383) for 2 hours at room temperature. After washing four times with PBS, the cells were incubated with Alexa-Fluor488 labeled Goat anti-Rabbit IgG (H+L) secondary antibody (Thermo Scientific, #A32731) for 1 hour at room temperature in a dark room. After washing four times with PBS, the cells were mounted with ProLong Gold with DAPI solution (Thermo Scientific, #3693).
3
Engineered Fluorescent Sec61β Constructs
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mEmerald-Sec61β and mCherry-Sec61β were acquired from Addgene (plasmids 54249 and 49155, respectively). GFP-KDEL was cloned as previously described (Merta et al., 2021 (link)). In brief, a pDsRed2-ER vector (632409; Clontech) was digested with AgeI and HindIII to remove DsRed2, and a PCR-amplified GFP sequence with AgeI and HindIII sites was ligated into the vector. SNAP-Sec61β and Halo-Sec61β were cloned as previously described (Bottanelli et al., 2016 (link)). Briefly, mEmerald-Sec61β (above) was digested with NheI and BgIII to remove mEmerald, and a PCR-amplified HaloTag or SNAP-tag sequence with NheI and BgIII sites was ligated in mEmerald’s place.
4
Subcellular Localization of FX Protein
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To determine the subcellular localization of the WT or mutant FX protein, a fluorescent antibody and two red fluorescent protein vectors that were designed to specifically label the endoplasmic reticulum and Golgi apparatus, respectively, were used. HEK293 cells grown on cover slips were transiently co-transfected with 1 μg of each plasmid constructs [pcDNA3.1(-), pcDNA/FX-WT, or pcDNA/FX-A275V] and 1 μg of pDsRed-Monomer-Golgi vector or pDsRed2-ER vector (Clontech Laboratories, Mountain View, CA, USA) using a PolyFect reagent. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed three times in PBS, permeabilized in 1% Triton X-100/ PBS, and blocked using 1% BSA in PBS. Monoclonal mouse antihuman FX antibody were incubated at 4 °C overnight, and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (H + L) (KPL,Gaithersburg, MD, USA) was added, followed by incubation for 1 h at room temperature. Then, the cover slips were mounted onto glass slides with DAPI/anti-fade solution (Millipore, MA, USA), followed by capturing fluorescence images using a confocal laser scanning microscope (Zeiss, LSM780, Germany).
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