Cells were seeded on glass slides overnight. After siRXR or siCtrl (40 nM) transfection for 24 h, and then Wnt3a (50 ng/mL) treatment for 6 h, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized using 0.5% Triton X‐100 for 20 min, and blocked using normal donkey serum for 30 min. The primary antibodies (β‐catenin, 1:200) were added and incubated at 4°C overnight. The slides were washed and incubated with Alexa Fluor 647‐conjugated secondary antibodies (1:100) at room temperature for 30 min, then DAPI (1 μg/mL) was used to stain nuclei. The images were taken under a Leica TCS SP5 II laser confocal microscope using the LSM‐510 confocal laser scanning microscope system, as previously described.17
Tcs sp5 2 laser confocal microscope
Manufactured by Leica
Sourced in Italy, Spain
The Leica TCS SP5 II is a laser confocal microscope designed for high-resolution imaging. It features a multi-channel detection system that allows for simultaneous capture of multiple fluorescence signals. The microscope is equipped with a range of laser lines to excite various fluorophores. The TCS SP5 II provides superior optical performance and advanced imaging capabilities for a variety of applications.
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Lab products found in correlation
5 protocols using tcs sp5 2 laser confocal microscope
1
Wnt3a-Induced β-Catenin Localization
2
Germline Transgene Experiments in C. elegans
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Germline transgene experiments were performed as described [68 ]. For Prbm-5::GFP transcriptional reporter, a transgene solution containing 20 ng/μl Prbm-5::GFP was injected to wild type. For Peft-3::rbm-5a cDNA::GFP reporter, a mixture containing 1 ng/μl of the transgene and 2.5 ng/μl of the pCFJ90 (Pmyo-2::mCherry) plasmid as co-injection marker was injected to wild type. For Punc-119::rbm-5a cDNA::GFP reporter, a mixture containing 0.2 ng/μl of the transgene and 2.5 ng/μl of pCFJ90 was injected to wild type. We tried but failed to obtain stable lines with a Prbm-5::rbm-5a cDNA transgene under control of the 1183-bp rbm-5 endogenous promoter at concentrations of 0.1, 1, 10 or 50 ng/μl in wild-type or double mutant backgrounds, suggesting that overexpressing rbm-5 using its endogenous promoter is highly toxic. Transgenic animals were observed using a Leica TCS SP5 II laser confocal microscope.
3
Trabectedin and Radiation-Induced DNA Damage
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Cells were treated as previously reported [31 (link)]. Briefly, cells were seeded on ibidi µ-Slide VI 0.4 in growth medium, then were starved with serum-free medium in the 24 h preceding irradiation. At this point, cells were pre-treated with trabectedin and/or irradiated at 4 Gy. The first samples were fixed 30 min after irradiation, and the remaining samples were incubated for 6 h and 24 h under standard cell culture conditions to allow repair of radiation-induced DNA double-strand breaks (DSBs). Then, cells were stained with the anti-gamma-H2AX antibody. A Leica TCS SP5-II laser confocal microscope (Leica, Milan, Italy) with a 40× objective was used to analyze the specimens. The mean immunofluorescent signal per nucleus emitted from gamma-H2AX was determined by the ImageJ software (NIH, Bethesda, MD, USA). The pan-nuclear signals without foci were excluded from the analysis. Then, the immunofluorescent signal resulting from each treatment is reported as the percentage of the immunofluorescent signal of the control cells.
4
Immunofluorescence Staining of RARα
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IF was performed as previously described.20 (link) After seeding cells on a glass slide and overnight culture, 4% paraformaldehyde was used to fix cells for 15 min. Triton X-100 (0.5%) was used for permeabilization of cells for 20 min. The blocked cells were incubated with 1:100 dilution of RARα -primary antibody. This was followed by 1:100 of secondary antibodies conjugated to Alexa Fluor 647. Nuclei were stained with 1 μg/mL DAPI. Leica TCS SP5 II laser confocal microscope (Leica, Barcelona, Spain) was used to record images.
5
Hydroxyapatite Nanoparticle Affinity Assay
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HAp disks with a diameter of 1 cm (100 mg/disk) prepared by compression moulding (FYD 40, Tianjin Sichuang Jingshi Technology Development Co., Ltd.) were used to evaluate the HAp affinity of nanoparticles. In brief, 5 mL dispersion of empty nanoparticles (1 mg mL -1 in normal saline, 5 mL) with or without Alen decoration was incubated with HAp disks in a brown vial for 0.5, 2 and 4 h in dark. The absorbance of dispersion was analyzed using a UV/vis spectrophotometer at 553 nm. The binding ratio to HAp was defined as the reduction in the percentage of absorbance at 553 nm.
The HAp disks were gently washed with water and then imaged using Image Station (Leica TCS SP5 II laser confocal microscope). Both nanoparticles were tested in triplicate.
The HAp disks were gently washed with water and then imaged using Image Station (Leica TCS SP5 II laser confocal microscope). Both nanoparticles were tested in triplicate.
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