The expression of TaRac6-GFPC and GFPN-TaRac6 were further confirmed by western blot. The total proteins of injected tobacco leaves were extracted using the Native lysis buffer (Solarbio, Beijing, China). Specifically, 10 μL PMSF (100 mM) and 10 μL protease inhibitor cocktail (EDTA-Free, 100 × in DMSO) were added per ml of lysate. The extraction of total protein and the western blot procedure used are described in Zhao et al. (2018) (link).
Lsm 510 confocal microscope
The LSM 510 confocal microscope is a high-resolution imaging system designed for advanced microscopy applications. It utilizes laser scanning technology to capture detailed, three-dimensional images of samples with high contrast and resolution.
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1 169 protocols using lsm 510 confocal microscope
Transient Expression of TaRac6 Fusion Proteins
The expression of TaRac6-GFPC and GFPN-TaRac6 were further confirmed by western blot. The total proteins of injected tobacco leaves were extracted using the Native lysis buffer (Solarbio, Beijing, China). Specifically, 10 μL PMSF (100 mM) and 10 μL protease inhibitor cocktail (EDTA-Free, 100 × in DMSO) were added per ml of lysate. The extraction of total protein and the western blot procedure used are described in Zhao et al. (2018) (link).
Immunofluorescence Staining for YAP and Cholesterol
Cholesterol staining was performed according to manufacturer’s protocol using the Cholesterol Cell-Based Detection Assay Kit (10009779) from Cayman Chemicals (Ann Arbor, Michigan, USA). Cells were permeabilized with PBS 0.5% Triton X-100 for 3 min before incubation with anti-GEF-H1 antibody (B4/7) for 1 h. Cells were washed three times before incubation with fluorescently labeled secondary antibody 1/250 in PBS for 1 h. Coverslips were mounted in mowiol and imaged on a Zeiss LSM510 confocal microscope.
Visualizing Erythrocytes and Giant Membrane Vesicles
Fluorescence Microscopy for Visualizing GFP and AmCyan Constructs
Immunocytochemistry and Cell Death Assays
To assess cell death, primary human RPE cells and ARPE-19 cells were fixed with methanol for 15 minutes, washed with 1 × PBS, then loaded with 2 μM Hoechst dissolved in a Locke’s solution (Promega) and incubated for another 15 minutes before imaging. Cells were then viewed by using a Zeiss LSM 510 confocal microscope under UV fluorescence. Images were recorded, and cell apoptosis was assessed by using an automated unbiased method70 (link).
Transient Transformation of Petunia Protoplasts
Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assay Yeast two-hybrid assays were performed as reported before (Quattrocchio et al., 2006) . In BiFC assays, plasmids encoding N-terminal fusions of protein of interest to nYFP and cYFP and, to mark the plasma membrane of transformed cells, RFP-SYP122 were transformed into protoplasts isolated from petunia M1xV30 (wild-type) petals. Transfected cells were imaged with a Zeiss LSM510 confocal microscope with a 40x/1.2 water objective at 24 hours after transformation. Over hundred transfected cells expressing RFP-SYP122 were observed for each independent experiment, and around 20 cells were imaged by confocal microscopy.
Duolink™ Protein Interaction Analysis
Live Imaging of Bacterial Colony Growth
Investigating Prostate Cancer Cell Lines
Immunofluorescence Microscopy Assay
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