Transfected HEK-293T cells grown in Cellvis plastic dishes were first fixed with 4% paraformaldehyde at room temperature for 30 min, then stained with DiD or ActinRed (diluted in 0.5% Triton X-100 PBS) for ∼30 min after PBS rinses. After washing off the fluorescent dye with PBS, fixed cells were embedded in DAPI-Fluoromount (Beyotime, Shanghai, China) and characterized with a Leica TCS SP8 imaging system in fluorescence imaging mode. The resulting images were analyzed with ImageJ (Java 1.8.0_172/1.52b) (Schindelin et al., 2012 (link)).
Dapi fluoromount
Manufactured by Beyotime
Sourced in China
DAPI-Fluoromount is a mounting medium used in fluorescence microscopy. It is designed to preserve fluorescent signals and provide a clear background for imaging. The product contains the DNA-binding dye DAPI, which emits blue fluorescence when bound to nucleic acids.
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Lab products found in correlation
3 protocols using dapi fluoromount
1
Fluorescent Imaging of Transfected Cells
2
Fluorescent Imaging of Transfected HEK Cells
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Transfected HEK-293T cells grown in Cellvis plastic dishes were firstly fixed with 4% paraformaldehyde at room temperatures for 30 min and then stained with DiD (diluted in 0.5% Triton X-100 PBS) for ∼30 min after PBS rinses. Fixed cells were embedded in DAPI-Fluoromount (Beyotime, Shanghai, China) after washing off the fluorescent dye with PBS, and characterized with a Leica TCS SP8 imaging system in fluorescence imaging mode. 405 Diode laser was used for DAPI detection; 488 Argon was used for GFP detection; DPSS 561 was used for mcherry detection; HeNeB 633 was used for DiD detection. The resulting images were analyzed with ImageJ (Java 1.8.0_172/1.52b) (Schindelin et al., 2012).
3
Lung Tissue Cytokine Expression Analysis
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A total of 8, 16 and 24 h following reperfusion, paraffin-embedded lung sections underwent IF staining of TNF-α and IL-10. Following routine de-paraffinization and rehydration, tissue sections were incubated with PBS containing 3% goat serum (Sigma, St. Louis, MO, USA) for 30 min at 37°C, and were then incubated overnight at 4°C with TNF-α (1:500, rabbit) and IL-10 (1:100, rabbit; catalog no. Ab9969) primary antibodies (both from Abcam, Cambridge, UK) which were diluted in PBS containing 2% normal goat serum. A negative control was performed by adding PBS instead of the primary antibody. Subsequently, sections were washed three times with PBS and were incubated with Cy3 fluorescence-labeled secondary antibody (1:200, anti-rabbit; catalog no. 111-165-003; Jackson Laboratory, Bar Harbor, ME, USA), in the dark for 30 min at 37°C. Sections were then washed three times with PBS, mounted onto gelatin-coated glass microscope slides, air dried and cover-slipped in a glycerol-based mounting medium. Cell nuclei were visualized by DAPI-Fluoromount (Beyotime Institute of Biotechnology, Shanghai, China). Photomicrographs were captured under a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
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