Fam-labeled circFAM73A and Cy3-labeled miR-490-3p probes were designed and synthesized by Servicebio (Wuhan, China). FISH experiments were performed according to the manufacturer’s protocols. Briefly, for the cell assay, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. For GC tissues, 4-mm thick sections were cut from paraffin-embedded blocks and then deparaffinized and rehydrated. Hybridization of cells or tissue was performed with specific probes in a dark moist chamber at 37 °C overnight. The slices were sealed with parafilm containing DAPI. Images were acquired by a Leica SP5 confocal microscope system (Leica Microsystems, Mannheim, Germany).
Sp5 confocal microscope system
Manufactured by Leica
Sourced in United Kingdom
The Leica SP5 confocal microscope system is a high-performance imaging platform designed for advanced microscopy applications. It features a modular design, allowing for customization and adaptation to diverse research needs. The SP5 employs confocal laser scanning technology to capture high-resolution, three-dimensional images of biological samples with exceptional clarity and detail.
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Lab products found in correlation
Blocking reagent, by Roche (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Citifluor, by Agar Scientific (3 mentions)
Goat α mouse alexa 594, by Thermo Fisher Scientific (2 mentions)
Glass slide, by Avantor (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Ix 71 widefield microscope, by IDEX Corporation (1 mentions)
Ab81371, by Abcam (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
17 protocols using sp5 confocal microscope system
1
FISH Assay for circFAM73A and miR-490-3p
2
Immunofluorescence Labeling of Cilia Proteins
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Cells grown on 0.17 mm thick (#1.5) coverslips (Fisher Scientific, Loughborough, UK) were washed twice in PBS, and then fixed in ice-cold methanol at −20°C for 5 min. For Smo labelling, cells were fixed for 10 min at room temperature (RT) in 4% paraformaldehyde (PFA) and permeabilised with PBS containing 0.1% Triton X-100 for 5 min. For DHC2 (DYNC2HC1) labelling, cells were permeabilised with PBS containing 0.3% Triton X-100 for 10 min. Subsequently, cells were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min. The coverslips were incubated with primary antibodies for 1 h, washed in PBS and then incubated with relevant secondary antibodies for 1 h. Nuclear staining was performed using DAPI (Life Technologies) at a concentration of 1 µg ml−1 in PBS for 3 min. Cells were then rinsed twice in PBS before mounting on glass slides (VWR) using Mowiol 4-88 mounting medium. Cells were imaged using an Olympus IX-71 widefield microscope with a 63× oil immersion objective (NA 1.4), and excitation and emission filter sets (Semrock, Rochester, NY) controlled by Volocity software (version 4.3, Perkin-Elmer, Seer Green, UK). For ciliary DYNC2HC1 (Fig. 5A,B ), cells were imaged on a Leica SP5 confocal microscope system (Leica Microsystems, Milton Keynes, UK). Images were acquired as 0.5 µm z-stacks.
3
Visualizing Sense RNA Foci using FISH
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To visualize sense RNA foci, RNA fluorescence in situ hybridization (FISH) was performed as described12 (link) using a 5′ TYE-563-labelled LNA (16-mer fluorescent)-incorporated DNA probe (Exiqon, Inc.). For human post mortem spinal cord tissue, the study was approved by the South Sheffield Research Ethics Committee and informed consent was obtained for all samples. RNA-FISH and immunohistochemistry were performed on formalin fixed paraffin-embedded (FFPE) tissues as in reference12 (link). Anti-SRSF1 antibody (Cell Signaling #8241) at a dilution of 1:200. Mounted slides were visualized using a Leica SP5 confocal microscope system and a 63/1.4 oil immersion objective lens. The presence of RNA foci was assessed at high resolution (848 μm2 per image, 393 × 393 pixels) using 0.9 μm z-stacks through the entire volume of the cell.
4
Quantifying RNA Foci and RBP Localization
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RNAfociand RBP: RNA foci were visualised using a Leica SP5 confocal microscope system with a X63/1.4 oil immersion objective lens. The presence of foci was assessed within a high-resolution (1433 mm2 per image, 511 × 511 pixels) z-stack made up of images at 0.13 µm intervals through the entire nuclear volume of the cell under consideration. RNA foci were quantified manually. The same imaging was used for RNA foci and RBP (SRSF1, SRSF2 and NCL) co-staining, and for each RBP foci co-stain 50 NSC34 (G4C2)102 cells were analysed for co-localisation.
NCL: NCL staining in the NSC34 cells and tissue was visualised using a Leica SP5 confocal microscope system with an X63/1.4 oil immersion objective lens. (3775 mm2 per image, 511 × 511 pixels) z-stack made up of images at 0.5-mm intervals through the entire nuclear volume of the cell under consideration. To quantify the NCL area relative to the nuclear area, we used the analysis previously described by Haeusler et al. 2014 (28 (link)). Briefly, a threshold of 50–100 was set in FIJI to measure the nucleolar NCL area, relative to the nuclear area (defined by DAPI staining). Data are means ± SD n = 3 for NSC34 cells.
TDP-43: TDP-43 staining in NSC34 cells were visualised using a Nikon DS Ri1 Eclipse.
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RNAfociand RBP: RNA foci were visualised using a Leica SP5 confocal microscope system with a X63/1.4 oil immersion objective lens. The presence of foci was assessed within a high-resolution (1433 mm2 per image, 511 × 511 pixels) z-stack made up of images at 0.13 µm intervals through the entire nuclear volume of the cell under consideration. RNA foci were quantified manually. The same imaging was used for RNA foci and RBP (SRSF1, SRSF2 and NCL) co-staining, and for each RBP foci co-stain 50 NSC34 (G4C2)102 cells were analysed for co-localisation.
NCL: NCL staining in the NSC34 cells and tissue was visualised using a Leica SP5 confocal microscope system with an X63/1.4 oil immersion objective lens. (3775 mm2 per image, 511 × 511 pixels) z-stack made up of images at 0.5-mm intervals through the entire nuclear volume of the cell under consideration. To quantify the NCL area relative to the nuclear area, we used the analysis previously described by Haeusler et al. 2014 (28 (link)). Briefly, a threshold of 50–100 was set in FIJI to measure the nucleolar NCL area, relative to the nuclear area (defined by DAPI staining). Data are means ± SD n = 3 for NSC34 cells.
TDP-43: TDP-43 staining in NSC34 cells were visualised using a Nikon DS Ri1 Eclipse.
5
Immunofluorescence Staining of Cultured Cells
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Cells were seeded on glass coverslips, and fixed 24 hours later for 10 minutes in 3.7% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2 pH 6.9) at 37°C. After washing with PBS, cells were permeabilized with 0.1% Triton-X100 for 10 minutes, washed with PBST, and blocked using TNB (100 mM TRIS pH 7.5, 150 mM NaCl, 0.5% Blocking Reagent (Roche)) for 30 minutes. Cells were incubated with primary antibody as indicated, in TNB for one hour. Subsequently cells were washed five times with PBST, and incubated with secondary antibodies (Goat α Mouse Alexa 594 (Invitrogen)) in TNB for one hour. Next, cells were washed five times with PBST and dehydrated using alcohol, prior to embedding them in Citifluor (Agar Scientific) containing 400 ng per μL DAPI (Sigma) and sealing the slides with nail varnish. Images were recorded on a Leica SP5 confocal microscope system using 488 nm and 561 nm lasers for excitation, a 63× lens for magnification, and were analyzed with Leica confocal software.
6
Immunofluorescent Staining of PML Bodies
7
Confocal Imaging of Nuclear Foci
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Primary visualization of foci was performed using a Leica SP5 confocal microscope system with a ×63/1.4 oil immersion objective lens. The presence of foci was assessed within a high resolution (1433 µm2 per image, 511 × 511 pixels) z-stack made up of images at 0.13-µm intervals through the entire nuclear volume of the cell under consideration.
8
Fluorescent Antibody Skin Biopsies
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Skin biopsies were performed 2 hours after the intradermal administration of fluorescently labeled antibodies, embedded in OCT and frozen in liquid nitrogen. Ten micrometer sections were fixed in 4% of PFA for 15 min and cell nucleus was stained with DAPI. Images were acquired with the Leica SP5 confocal microscope system.
9
RNA Foci Visualization via LNA Probe
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A 5′ TYE-563-labelled LNA (16-mer fluorescent)-incorporated DNA probe was used against the sense (Exiqon, Inc.; batch number 607323) and the antisense RNA hexanucleotide repeat (Exiqon, Inc.; batch number 610331). Slides were prepared and RNA foci were visualised as described previously [3 (link)] using a Leica SP5 confocal microscope system with a ×63/1.4 oil immersion objective lens. Briefly prehybridisation was followed by overnight hybridization at 66 °C in a humid atmosphere. A single wash at room temperature with 2 × SSC/0.1 % Tween-20 preceded three washes at 65 °C with 0.1 × SSC. Slides were then mounted in DAPI Vectashield or processed further for dual staining of RNA and protein.
10
Optogenetic Translocation of Cellular Proteins
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Activation of NLS-mCherry-LEXY translocation in single cells (Fig. 1f,g ) was performed using a Leica Sp5 confocal microscope system equipped with automated temperature (37 °C) and CO2 (5%) control, a multiline argon laser and a PL Apo CS × 40 oil objective (numerical aperture=1.3). Cells were focused using the mCherry signal activated with the 561-nm laser line and a circular region of interest (ROI; ∼30 μm2) was placed onto single, selected cells. The ROI was scanned with a 458-nm laser beam (∼2 μW intensity) for 30 ms every 10 s for 10 min following a 20-min dark-recovery phase. mCherry was imaged in parallel every 10 s for 30 min using the 561 laser line for excitation. Laser intensity was measured with a Laser Power and Energy meter (Nova). p53-mCherry-LEXY translocation (Fig. 4b,c ) was induced by irradiating a whole field of view with a 458 nm laser beam (intensity ∼2 μW) every 30 s for 40 min following a 40-min dark-recovery phase. In parallel, mCherry images were taken every 30 s during the blue light induction and every 5 min during the dark-recovery phase.
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