The direct observation of Upf2-EJC complex formation was performed using a Duolink® kit (Olink Bioscience, Uppsala, Sweden; product now owned by Sigma-Aldrich) following the manufacturer's protocol (23 (link)). This method enables the visualization of complex formation in cells by proximity ligation of single-stranded DNA conjugated with a secondary antibody (24 (link)), and is available for the complex in nuclei and cytoplasm (25 (link)). Signal can be observed when the distance between the secondary antibodies is <40 nm. Therefore, this method can detect not only direct protein-protein interactions, but also complex formation, assuming that the bound first antibodies are proximal enough. The first antibodies used in the present study were as described in the above Immunostaining and observation section. Images were captured with either an Axiovert 200 M inverted fluorescence microscope or an LSM 710 confocal point-scanning microscope (Carl Zeiss, München, Germany).
Lsm 710 confocal point scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 710 is a confocal point-scanning microscope developed by Zeiss. It is designed for high-resolution imaging of fluorescently labeled samples. The LSM 710 uses a focused laser beam to excite fluorophores within the sample, and a pinhole is used to reject out-of-focus light, enabling the acquisition of optical sections with improved contrast and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m inverted fluorescence microscope, by Zeiss (1 mentions)
Duolink kit, by Olink (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
4 protocols using lsm 710 confocal point scanning microscope
1
Visualizing Upf2-EJC Complex Formation
2
Multicolor Immunofluorescence Analysis
3
Immunostaining of Plasmodium Parasites in Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Livers isolated from infected mice were fixed with 4% paraformaldehyde at room temperature for 2 h. The fixed liver lobes were cut into 50-μm-thick sections using the Vibratome VT 1000S (Leica). Following blocking in 2% bovine serum albumin and 0.3% Triton X-100 at 4 °C overnight, liver sections were stained with goat anti-P. berghei UIS4 (1:1,000) (ref. 57 (link)) and mouse anti-P. berghei HSP70 (1:1,000) (ref. 58 (link)). The secondary antibodies used for detection were: Alexa Fluor 555 donkey anti-goat antibody and donkey anti-mouse conjugated to Alexa Fluor 488 (all 1:500). Cell nuclei were stained with diamidino-2-phenylindole. Stained liver sections were mounted on microscope slides with Fluoromount-G (SouthernBiotech). Images were acquired on a LSM 710 confocal point-scanning microscope (Zeiss).
4
Immunofluorescence Imaging of Dystrophin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For microscopy analysis, cells were cultured onto 0.1% gelatin-coated glass coverslips (10x10 mm2, Normax). Cells were first fixed with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in phosphate-buffered saline (PBS) for 10 min at room temperature, and then permeabilized with 0.5%Triton X-100 in PBS for 10 min at room temperature. Next, cells were incubated for 30 min at room temperature in 1%BSA and 0.05%Tween20 in PBS. Cells were then incubated with polyclonal rabbit anti-dystrophin antibody (Abcam ab85302, dilution 1:100) for 60 min at room temperature followed by incubation with tetramethylrhodamine (TRITC) conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch; dilution 1:200) for 60 min. Antibodies were diluted in 1%BSA, 0.05%Tween20 in PBS. To counterstain the nuclei, cells were incubated for 10 min with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Sigma). VECTASHIELD® Antifade Mounting medium (Vector Laboratories) was used. Cells were imaged with a LSM 710 Confocal Point-Scanning Microscope (ZEISS), using the lasers Diode 405–30 (405 nm) and DPSS 561–10 (561 nm).
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!