Eighty thousand A549 cells were seeded on coverslips. On the following day, cells were incubated with ca. 80 bound particles of each virus on ice as described above. Inocula were removed, and cells were incubated with infection medium at 37°C. Cells were fixed, quenched, and permeabilized. Samples were stained with mouse anti–hexon 9C12 and goat anti-mouse Alexa Fluor 488 in blocking buffer containing DAPI. Cells were treated with succinimidyl ester Alexa Fluor 647 and mounted onto glass holders. Samples were imaged with a Leica SP8 upright confocal microscope. For image analysis, virus particles were segmented according to their hexon signal and masked with the nuclear signal.
Sp8 upright confocal microscope
Manufactured by Leica camera
The SP8 upright confocal microscope is a high-performance imaging system designed for advanced biological and materials science research. It features a modular architecture, allowing for customization to meet specific experimental needs. The SP8 utilizes laser-based excitation and precise optical detection to capture detailed images of samples with exceptional clarity and resolution.
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Lab products found in correlation
Alexa fluor 633 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Dako aqueous mounting medium, by Agilent Technologies (2 mentions)
Alexa fluor 555 conjugate, by Thermo Fisher Scientific (2 mentions)
C fl b 2e c, by Nikon (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Cf633 conjugated wheat germ agglutinin, by Biotium (2 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Rabbit anti neun, by Merck Group (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Leica application suite x imaging software, by Leica camera (1 mentions)
Rat anti cd68, by Abcam (1 mentions)
Sudan black b, by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Dapi containing mounting medium, by Vector Laboratories (1 mentions)
Leica cm1860 cryostat, by Leica camera (1 mentions)
Paxillin, by Abcam (1 mentions)
Phospho histone h3 ser10, by Merck Group (1 mentions)
Vinculin, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
23 protocols using sp8 upright confocal microscope
1
Quantifying Virus Particle Binding
2
Quantifying Actin Filament Intensity
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Cells grown on coverslips and submitted to different treatments were rinsed with PHEM buffer (60 mM PIPES, 25 mM HEPES pH 6.9, 5 mM EGTA and 2 mM Mg acetate), pre-lysed in PHEM buffer containing 0.5% Triton X-100 and fixed in PHEM buffer containing 3.7% paraformaldehyde/0.02% glutaraldehyde. Autofluorescence of paraformaldehyde was diminished by incubation of cells in NH4Cl 50 mM/PBS pH 7.4 for 10 min. Cells were permeabilized with 0.1% Triton X-100 (1% BSA/PBS) for 5 min at room temperature and rinsed with PBS. To localize actin filaments, cells were incubated with rhodamine–phalloidin (Invitrogen) for 20 min at room temperature in a humid chamber at concentrations according to the manufacturer’s protocol. The coverslips were rinsed in PBS and mounted on the slide using Prolong Gold Antifade Mountant (Invitrogen). Images were obtained using a Leica SP8 upright confocal microscope. Z-stacks (about 15 images taken at different z-planes) encompassing the entire volume of the cells were taken. The merged z-stacks were obtained using the ImageJ software. Actin filament intensity per cell area was determined indirectly by measuring the intensity of fluorescence (phalloidin staining) using the ImageJ software, and more than 30 cells per sample were measured.
3
Immunofluorescent Staining of Mouse Brain
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After euthanasia, the mouse was perfused with 0.25 mg/mL heparin in ice-cold PBS. Then, the mouse was perfused with ice-cold 4% paraformaldehyde (PFA). The brain was dissected and transferred to 4% PFA for 2 hours on ice. The brains were transferred to 20% sucrose in PBS and incubated overnight at 4°C. The next day, the brains were transferred to 30% sucrose in PBS and incubated overnight at 4°C. The brains were embedded in OCT and 10 μm sections were cut. The brain sections were washed 2× in TBS and blocked in 5% normal goat serum in TBST for 1 hour at room temperature. The brains were stained overnight at 4°C with HER2 (Santa Cruz sc-33684, 1:250). The sections were washed 3× with TBST and incubated with secondary antibody Alexa Fluor 633 Goat Anti Mouse IgG (H + L) (Thermo Fisher, 1:250) for 1 hour in the dark at room temperature. The slides were washed 3× with TBST. The sections were stained with Hoescht 33342 (5 μg/mL) for 10 minutes at room temperature in the dark. The sections were washed 3× with TBST and 1× with TBS. The cover slips were mounted using Dako aqueous mounting medium (Dako). The sections were imaged using a Leica SP8 upright confocal microscope.
4
Visualizing Microglia and Neurons
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To visualize microglia and neurons, sections (Bregma - 1.5 mm) were incubated with rabbit anti-Iba1 (1:1000; Wako) or rabbit anti-NeuN (1:1000; Millipore) primary antibody, respectively, followed by Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (1:500, Invitrogen). In order to visualize microglial lysosomes, sections were incubated with rabbit anti-Iba1 (1:1000; Wako) and rat anti-CD68 (1:500; Abcam) primary antibodies, Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 647 donkey anti-rat IgG secondary antibodies (1:500, Invitrogen), and 0.1% Sudan Black B (Sigma-Aldrich) solution in 70% ethanol for 2 min prior to cover-slipping. Slides were then imaged using a Leica SP8 upright confocal microscope at 63X magnification and sequential optical sections captured using the Leica Application Suite X imaging software. Lipofuscin was imaged at 488 nm excitation and 495–545 nm emission. Sequential optical sections were analyzed using ImageJ software (NIH). For each animal, 2–4 images were quantified and averaged, and these values were used to calculate the mean and standard error for each experimental group.
5
Fluorescent Labeling and Imaging of Cells
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After the CuAAC reaction, the cells were fixed with 2% formaldehyde in PBS for 30 min at room temperature. After fixation, all samples were incubated in 20 μg mL–1 avidin–FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS for six times and their wide-field fluorescence images were captured with a Nikon Eclipse 80i microscope in bright field and fluorescence modes using a 10× objective, a FITC emission filter (Nikon C-FL B-2E/C, 465–495 nm) and 400 ms of exposure time. For confocal imaging, the fixed cells were first incubated in 3 μg mL–1 wheat germ agglutinin, Alexa Fluor 555 conjugate (Life Technologies) in PBS for 10 minutes for staining the plasma membrane. Samples were washed with PBS for 3 times and incubated in 20 μg mL–1 avidin–FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS six times and mounted on glass slide with DAPI containing mounting medium (VECTOR Laboratories). The samples were imaged with a Leica SP8 upright confocal microscope using a 63× objective and the following excitation wavelengths: 405 nm (DAPI), 496 nm (FITC), 561 nm (Alexa 555).
6
Longitudinal Imaging of Arabidopsis SAM
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Z-stacks of biological quadruplicate wild-type Arabidopsis Colombia (Arabidopsis thaliana Colombia) and katanin1 (Bichet et al., 2001 (link)) shoot apical meristems (SAM) carrying YFP targeted to the plasma membrane (Yang et al., 2016 (link)) were imaged at several timepoints, 0 h (T0), 11 h (T1) and 22 h (T2). Confocal imaging was performed on a Leica SP8 up-right confocal microscope using long working-distance water immersion objectives (AP 40x/0.8). Excitation of YFP was performed using an argon laser at 514 nm. Images were collected at 529–545 nm for YFP using HyD detector. Time-lapse imaging was performed as described previously (Kierzkowski et al., 2012 (link)). Data were collected as 12-bit images and prepared and analyzed using MorphoGraphX software (de Reuille et al., 2015 ).
7
Immunohistochemical Analysis of VTA and Striatal Sections
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Mice were anesthetized by ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and transcardially perfused with 0.1 M sodium phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 4% sucrose-PBS (pH 7.4). After perfusion, the brain was removed and post-fixed in the same fixative for 4 hr at 4°C, and was dehydrated in increasing concentrations of sucrose (20% and 30%) in 0.1 M PBS at 4°C. The fixed brain was frozen on dry ice, and coronal VTA or striatal sections (20 µm) were cut with a Leica cryostat (CM1860). VTA sections were incubated with primary antibody against tyrosine hydroxylase (TH, rabbit polyclonal, 1:300, Santa Cruz Biotechnology, Inc, Dallas, TX) at 4°C for 48 hr. After rinsing with PBS three times at 15 min each, VTA sections were then incubated in secondary antibody: Goat anti-rabbit IgG Alexa Fluor-488 for 4 hr at room temperature in the dark. Coronal striatal sections were rehydrated in PBS and permeabilized with 0.1% Triton X-100 in PBS followed by 640/660 deep-red fluorescent Nissl stain (N-21483) incubation for 20 min. Confocal imaging was performed using a Leica SP8 upright confocal microscope.
8
Imaging and Analysis of Arabidopsis Seeds
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The samples were prepared as described in the previous sections. Z-stacks of Col-0 and iku2 seeds expressing pELA1:3X-VENUS-N7 were acquired using a Leica SP8 upright confocal microscope equipped with a 40x water immersion objective (HCX APO L UV 40×/0.8 W). After imaging, the water was removed and replaced with a drop of clearing solution to allow subsequent embryo staging (see clearing section). Nuclear Fluorescence intensities were measured using a custom-made macro script developed in ImageJ where the nuclei were segmented using on z-stack projections (Sum-slices) using a marker-based watershed (https://imagej.net/Marker-controlled_Watershed ).
9
Immunofluorescent Staining of Mouse Brain
10
Immunofluorescence Imaging of Fibroblasts
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Fibroblasts were grown on 24 well plates with glass coverslips in each well. After 4 h of BCM treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized 10 min with 0.1% Triton X-100 (Sigma) and 30 min blocking with 0.1 M L-lysine monohydrochloride in PBS. Samples were incubated with Cx43 (Sigma C6219, 1:4000), ZO-1 (Invitrogen 339 100, 1:100), β-catenin (Abcam ab6302, 1:2000), α-tubulin (Sigma T6074, 1:1000), GM130 (BD transduction Laboratories 610 822, 1:200), phospho-Histone H3 (Ser10) (Milipore 09–797, 1:1000), vinculin (Sigma) and paxillin (Abcam) at room temperature for 1 h. Samples were washed with PBS, incubated with secondary antibodies for 1 h at room temperature, stained with DAPI and/or rhodamine-phalloidin, washed in PBS and mounted in Citifluor® (Glycerol/PBS solution, Citifluor Ltd). Immunofluorescence was imaged on a Leica SP8 upright confocal microscope. Optimal gain and offset were set and kept constant during image acquisition. Minimum of three regions were imaged of each biological replicate.
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