Human fetal RPE cells were fixed for 20 minutes at RT in the fixation buffer Cytofix (BD Bioscience, San Jose, CA, USA), permeabilized in 0.2% TritonX (Sigma-Aldrich) for 20 minutes at RT and blocked with 1% bovine serum albumin (Sigma-Aldrich) in 0.2% TritonX for 30 minutes at RT. After staining the samples against the tight junction protein ZO-1 for 24 hours at 4°C and secondary antibody Cy5 (Thermo Fisher Scientific) for one hour at RT, NucBlue (Molecular Probes, Eugene, OR, USA) were added for nuclei staining 20 minutes before imaging with the Leica TCS SP8 confocal laser microscope (Leica Microsystems, Mannheim, Germany).
Leica tcs sp8 laser confocal microscope
Manufactured by Leica Microsystems
Sourced in Germany
The Leica TCS SP8 is a laser confocal microscope that allows for high-resolution, three-dimensional imaging of samples. It utilizes a number of laser lines to excite fluorophores within the sample, enabling the capture of detailed, optical sections. The microscope is designed to provide precise control over the illumination and detection of signals.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cytofix, by BD (1 mentions)
Ultramicrotome, by Leica Biosystems (1 mentions)
Spurr resin, by Electron Microscopy Sciences (1 mentions)
Polylysine coated glass slides, by Merck Group (1 mentions)
Antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ab150119, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab108209, by Abcam (1 mentions)
Ab150081, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Mab161, by R&D Systems (1 mentions)
Uv 3600 uv vis spectrometer, by Shimadzu (1 mentions)
20 mm glass bottom dish, by Cellvis (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Anti ev d68 vp1 antibody, by GeneTex (1 mentions)
14 protocols using leica tcs sp8 laser confocal microscope
1
Immunostaining of Fetal RPE Cells
2
Apoptosis Evaluation in Cochlear Explants
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To assess apoptosis in cultured cochlear explants, DNA fragmentation was evaluated by the TUNEL assay. After processing, the explants underwent incubation for 30 min at 37°C shielded from light according to the TUNEL staining instructions (Roche, Cat.no. 11684795910). Images of all cochlear explants were acquired under a Leica TCS SP8 confocal laser microscope (Leica Microsystems).
3
Quantifying Nuclear Translocation in TJ356 Worms
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Approximately 45 randomly selected TJ356 worms from each experiment were anesthetized after 6 days of culture. All fluorescence determinations were performed with a Leica TCS SP8 confocal laser microscope (Leica Microsystems, Buffalo Grove, IL, USA) using a 10x objective lens. Images were acquired with a 488 nm excitation filter and a 500/525 nm emission filter. TJ356 worms were classified as cytoplasmic, intermediate cytoplasmic/nuclear, and strong nuclear translocation. Three independent experiments were performed.
4
Transient Expression of AhCML69 in N. benthamiana
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Transient expression of AhCML69 in N. benthamiana was performed according to the aforementioned protocol. Two days later, the infiltrated leaves were cut and observed under a Leica TCS SP8 confocal laser microscope (Leica Microsystems (Shanghai) Trading Co., Ltd., Mannheim, Germany). A wavelength of 488 nm was used for GFP excitation, and the 510 nm wavelength of the emission signal was obtained for the GFP channel. A wavelength of 640 nm was used for chlorophyll excitation, and 675 nm for the emission signal was used for the chlorophyll channel.
5
Casparian Band Visualization in Chinese Fir
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Primary roots of 6-week-old Chinese fir plants, which were approximately 8-10 cm long, were divided into three zones: zone I (0-1 cm), zone II (1-4 cm) and zone III (4-8 cm), and 0.3-cm segments were cut at 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 cm from the root tip. The samples were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 24 h at 4 °C. The samples were then rinsed in 0.1 M cacodylate buffer, dehydrated in a graded ethanol and acetone series, then infiltrated and embedded in Spurr resin (Electron Microscopy Sciences). The resin blocks were polymerized for 24 h at 70 °C, then cut into 10-µm slices using an ultramicrotome (Leica Biosystems). The Casparian bands were stained with 0.1% (w/v) berberine hydrochloride for 1 h and counter-stained with 0.5% (w/v) aniline blue for an additional hour, as reported previously (Brundrett et al. 1988; Man et al. 2018) . The stained sections were viewed under a Leica TCS SP8 confocal laser microscope (Leica Microsystems) with an excitation wavelength of 488 nm and a detection wavelength of 520 nm.
6
Immunofluorescence Analysis of NF-κB/p65
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Immunofluorescence analysis of NF-κB/p65 was conducted as previously described.44 (link) In brief, 2 × 105 cells were seeded into a 35 mm glass bottom SPL confocal dish (SPL Life Sciences, Gyeonggi-do, Korean)overnight. They were pretreated with TTN (4 μg/ml) for 1 h, stimulated with LPS (1 μg/ml) for another hour, and then fixed and stained with Hoechst3342 (1 μM). Images were taken under a Leica TCS SP8 laser confocal microscope (Leica Microsystem, Wetzlar, Germany) using × 60 magnification with excitation and emission wavelengths at 588 and 615–690 nm, respectively.
7
Quantifying Protein Aggregation in Cells
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Cells were grown to mid-log phase (OD600 = 0.6) in SD medium supplemented with 2 mg/L adenine and the indicated concentrations of the chemical chaperones. One milliliter of cells was washed with PBS, sonicated for 2 min at 43 kHz (Ultrasonic Cleaner 0.6L 220V DG-1, MRC Labs, Israel) and resuspended in 50 µL of ProteoStat (prepared according to the manufacturer’s instructions) diluted 1:250 in PBS. Cells were incubated in the dark for 15 min at room temperature. 10 µL of each sample was deposited on polylysine-coated glass slides (Sigma-Aldrich). Cells were imaged using Leica TCS SP8 laser confocal microscope with ×100 1.4 NA oil objectives (Leica Microsystems, Wetzlar, Germany). Excitation and emission wavelengths of 488 nm and 500–600 nm, respectively. The results displayed are representative of three independent biological repeats.
8
Fluorescent Visualization of Autophagy
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HK-2 cells were transfected with pDSRed-LC3 the lentivirus (MOI, 20) for 48 h and then fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, washed three times with PBS for a total of 10 min each and permeabilized with pre-chilled (−20°C) 70% ethanol for 20 min. The cells were then blocked with 8% BSA diluted in PBS for 1 h and washed with PBS. Following incubation for 2 h, transfected HK-2 cells were labeled with DAPI (5 mg/l) and incubated with pDSRed-LC3 (cat. no. 38171200626; purchased from REBIO; Shanghai Shengwu Gongcheng Co., Ltd.) overnight at 4°C. Subsequently, the treated cells were washed three times with PBS and incubated at room temperature with AlexaFluor® 488-conjugated anti-rabbit secondary antibody (dilution 1:1,000; cat. no. A-11008; Thermo Fisher Scientific, Inc) for 1 h. The cells attached to glass slides were removed from the 24-well plates in the dark room and placed faced up on a blotting paper, where a drop of antifade mounting medium (Invitrogen; Thermo Fisher Scientific, Inc.) was added. After the cells were dried, they were imaged using a Leica TCS SP8 laser confocal microscope (Leica Microsystems, Inc.).
9
Confocal Microscopy Imaging Protocol
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The specimens were scanned hierarchically with an interval of 0.5 μm/layer by a ×63 magnification oil-immersion objective mounted on a Leica TCS SP8 laser confocal microscope (Leica Microsystems, Wetzlar, Germany). The final images were superimposed. The optimal excitation wavelengths of 568 nm (red), 647 nm (far-red), and 358 nm (blue) for DAPI were used.
10
Immunofluorescent Imaging of ACE2, DC-SIGN, and SARS-CoV-2 Spike
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Cells were fixed and blocked with 5% BSA, and then incubated overnight at 4°C with rabbit anti-human ACE2 monoclonal antibody (ab108209; Abcam, UK), mouse anti-DC-SIGN monoclonal antibody (MAB161, R&D Systems, USA), mouse anti-L-SIGN monoclonal antibody (MAB162, R&D Systems, USA), or rabbit anti-SARS-CoV-2 spike monoclonal antibody (40150-R007, Sino Biological, China), depending on the experimental design. The cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (ab150113; Abcam, UK), 488-conjugated goat anti-rabbit IgG (ab150081; Abcam, UK), Alexa Fluor 647-conjugated goat anti-mouse IgG (ab150119; Abcam, UK) or Alexa Fluor 647-conjugated goat anti-rabbit IgG (ab150075; Abcam, UK) for 1 h at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, China). Images were obtained with a Leica TCS SP8 laser confocal microscope (Leica Microsystems, Germany).
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