HCT116 control, claudin-7 negative control and claudin-7 knockdown cells grown in confocal chambers (Nest 801006) were fixed in 4% paraformaldehyde for 20–30 minutes at room temperature and washed with PBS for 10 minutes before being blocked in 1% BSA for 60 minutes at room temperature. After blocking, the cells were incubated with primary antibodies. All antibodies were diluted in PBS containing 1% BSA. After washing, the cells were incubated with the corresponding secondary antibodies for 60 minutes at room temperature in the dark. A drop of DAPI was added to each well of the confocal chambers, and the chambers were kept at room temperature for 5 minutes in the dark. The samples were imaged using a laser confocal microscope, Nikon A1 (Nikon Corporation, Tokyo Japan).
Confocal chambers
Manufactured by NEST Biotechnology
Confocal chambers are specialized laboratory equipment designed for high-resolution optical imaging of biological samples. They utilize a focused laser beam and a pinhole aperture to selectively illuminate and detect light from a specific focal plane within a specimen, enabling the capture of detailed images with improved contrast and depth of field.
Automatically generated - may contain errors
2 protocols using confocal chambers
1
Immunostaining of Claudin-7 in Cell Lines
2
Mitochondrial Imaging and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated on confocal chambers (NEST) at a density of 35,000 cells per well. Next, cells were stained with Hoechst 33,342 (0.2 μg/ml, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min, following incubation, 50 nM MitoTracker Green FM (M7514, Thermo Fisher Scientific) or 25 nM tetramethylrhodamine methyl ester (TMRM) (I34361, Thermo Fisher Scientific) was added, for 30 min at 37 °C, in a CO2 incubator, then washed three times with PBS. After washing, the images of fluorescently labelled mitochondria and nuclei were captured using Leica TCS SP8 STED confocal laser scanning microscope (Leica). The relative intensities of Mito Tracker Green FM and TMRM were calculated in each cell using ImageJ software as previously described [50 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!