The cells were dropped to the cover glass in each well of six-well plates. After cultured for the desired time, the cells were fixed by 4% paraformaldehyde for 30 min, permeabilized by 0.2% TritonX-100 reagent for 2–5 min, blocked by 10% BSA (bovine serum albumin) diluted in PBS for 30 min, and then incubated overnight with the primary antibody against p50 (Santa Cruz, sc-8414, dilution 1:200) or p65 (Cell Signaling Technology, #8242, dilution 1:400) diluted in PBS containing 1% BSA. After washing with PBS, the slides were incubated with secondary antibodies conjugated with fluorescein isothiocyanate and rhodamine for 1 h in the dark room. Finally, after washing with PBS, the slides were stained with DAPI (Sigma-Aldrich) and anti-fade reagent. Then the protein expression was observed and photographed by using Leica DM14000B confocal laser scanning microscope.
Dm14000b confocal laser scanning microscope
Manufactured by Leica
The DM14000B is a confocal laser scanning microscope manufactured by Leica. It is designed to capture high-resolution, three-dimensional images of samples through optical sectioning. The microscope uses a laser as the illumination source and a pinhole to reject out-of-focus light, enabling the acquisition of sharp, detailed images.
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Lab products found in correlation
3 protocols using dm14000b confocal laser scanning microscope
1
Immunofluorescence Analysis of NF-κB Subunits
2
Immunofluorescence Assay for Apoptosis Markers
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A375 and SK-mel-28 cells were incubated on chamber slides in 6-well plates with indicated treatment, cells fixed for 10 min at room temperature (RT) with 4% paraformaldehyde, and then permeabilized with PBST (PBS with 0.2%Triton X-100), blocked with bovine serum albumin (BSA) 30 min and incubated with Cytochrome-c, or p65 or p50 antibodies (1:200 dilution) for overnight at 4 °C. Following 10-min washes for three times with PBS, cells were incubated with the fluorescein isothiocyanate- and rhodamineconjugated secondary antibodies for 30 min. Subsequently, the nuclei of stained samples were mounted with Vectashield solution containing 4′6-diamidino-2-phenylindole (DAPI). After five additional 10-min washes, the results were visualized by Leica DM 14000B confocal laser scanning microscope.
3
Analyzing Apoptosis and β-Catenin Localization
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A549 and H460 cells transfected with β-catenin siRNA were analyzed by flow cytometry to detect apoptosis. After 48 h transfection, the cells were stained with Annexin V-FITC and propidium iodide (PI) according to the instruction of Annexin V-PI Apoptosis Detection kit (Keygen, Jiangsu, China).
Confocal immunofluorescence HLF, H1299, A549 and H460 cells were seeded on chamber slides in 6-well plates. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with PBST, then blocked with 10% BSA for 30 min and incubated with antibodies against β-catenin (diluted to 1:100 with 5% BSA, CST 8480S) and CBP (diluted to 1:100 with 5% BSA, Santa Cruz sc-7300) overnight at 4°C. After washing 3 times, cells were incubated with the fluorescein isothiocyanate and rhodamine-conjugaed secondary antibodies (diluted to 1:200 with 5% BSA, CST 4408S and 4413S) for 1 h. Then the nuclei were stained with DAPI for 5 min, washed with PBS for 5 times. The images were captured by Leica DM14000B confocal laser scanning microscope.
Confocal immunofluorescence HLF, H1299, A549 and H460 cells were seeded on chamber slides in 6-well plates. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with PBST, then blocked with 10% BSA for 30 min and incubated with antibodies against β-catenin (diluted to 1:100 with 5% BSA, CST 8480S) and CBP (diluted to 1:100 with 5% BSA, Santa Cruz sc-7300) overnight at 4°C. After washing 3 times, cells were incubated with the fluorescein isothiocyanate and rhodamine-conjugaed secondary antibodies (diluted to 1:200 with 5% BSA, CST 4408S and 4413S) for 1 h. Then the nuclei were stained with DAPI for 5 min, washed with PBS for 5 times. The images were captured by Leica DM14000B confocal laser scanning microscope.
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