For immunofluorescence staining, cells grown on glass-bottom cell culture dishes were fixed with 4% paraformaldehyde at room temperature for 30 min. The samples were permeabilized with PBST (0.2% Triton X-100) at room temperature for 30 min. Then, the cells were blocked with 5% BSA at room temperature for 1 h. Then, the samples were incubated overnight with primary antibodies at 4 °C. Furthermore, the samples were washed three times with PBST and incubated at room temperature for 1 h with secondary antibodies and Hoechst 33342 (Invitrogen, H3570). Then, the samples were washed in PBST three times at room temperature and stored in the dark. The images were captured using a Zeiss-LSM880 inverted microscope. Data were processed using Zeiss software and ImageJ software.
Lsm 880 inverted microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 880 inverted microscope is a high-performance imaging system designed for advanced live-cell and super-resolution microscopy applications. It features a modular design that allows for customization to meet specific research requirements. The LSM 880 utilizes a sensitive detector system and advanced optics to capture detailed images of samples.
Automatically generated - may contain errors
Lab products found in correlation
Zen black software, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm t pmt detector, by Zeiss (2 mentions)
Zen software, by Zeiss (2 mentions)
Zen 2.3 lite blue edition, by Zeiss (2 mentions)
Eos 500d slr digital camera, by Canon (2 mentions)
Nucblue fixed cell readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Cx41 microscope, by Olympus (2 mentions)
Uis 2 20x 0.45 0 2 fn22 lens, by Olympus (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Oil immersion objective, by Zeiss (1 mentions)
Alexa fluor 488 conjugated neuronal class 3 β tubulin mouse monoclonal antibody, by Merck Group (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Fish kit, by RiboBio (1 mentions)
Bodipy 558 568 c12 dye, by Thermo Fisher Scientific (1 mentions)
Anti pi4p, by Echelon Biosciences (1 mentions)
Adeasy xl adenoviral vector system, by Agilent Technologies (1 mentions)
25 protocols using lsm 880 inverted microscope
1
Immunofluorescence Staining Protocol
2
Visualization and analysis of clot fiber density
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Clot fibrin fiber density was analysed using LSCM as described previously [16] (link). In short, plasma was spiked with a final concentration of 50 µg/mL AlexaFluor488-labeled fibrinogen and diluted 1:4 in TBS.
Clotting was initiated with the addition of 0.1 U/mL of human α-thrombin and 10 mmol/L of CaCl2 (final concentrations) and immediately transferred to six-chamber Ibidi slides. Clots were allowed to form for 2-4 h in a dark humidity chamber at room temperature after which they were imaged with a Zeiss LSM880 inverted microscope with a 40✕oil immersion objective (Carl Zeiss, Ltd., Cambridge, UK). Three micrographs were taken for each clot and Z-stacks (30 slices, 20 µm total distance) were combined and flattened at maximum intensity (ImageJ, NIH, Bethesda, MD, USA).
Clotting was initiated with the addition of 0.1 U/mL of human α-thrombin and 10 mmol/L of CaCl2 (final concentrations) and immediately transferred to six-chamber Ibidi slides. Clots were allowed to form for 2-4 h in a dark humidity chamber at room temperature after which they were imaged with a Zeiss LSM880 inverted microscope with a 40✕oil immersion objective (Carl Zeiss, Ltd., Cambridge, UK). Three micrographs were taken for each clot and Z-stacks (30 slices, 20 µm total distance) were combined and flattened at maximum intensity (ImageJ, NIH, Bethesda, MD, USA).
3
Corneal Whole-Mount Immunofluorescence Staining
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Frozen sections (7 µm) were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin at room temperature. The samples were incubated with primary antibodies overnight at 4°C and subsequently with corresponding secondary antibodies incubate for 1 hour at room temperature. All samples were counterstained with 4′,6-diamidino-2-Phenylindole (Sigma-Aldrich), and images were captured with an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan). Corneal whole-mount immunofluorescence staining was performed as previously described.24 (link) Mouse eyeballs were collected and fixed in Zamboni's fixative for 1 hour, then the cornea was dissected around the scleral–limbal region and blocked by phosphate-buffered saline with 0.1% Triton X-100, 2% goat serum, and 2% bovine serum albumin for 2 hours, and subsequently incubated in the same incubation buffer with Alexa Fluor 488 conjugated neuronal class III β-tubulin mouse monoclonal antibody (Merck-Millipore, Darmstadt, Germany) overnight at 4°C. After washing for five times, the flat mounts were imaged on an LSM880 Zeiss inverted microscope (Carl Zeiss Meditec, Jena, Germany). The quantification of corneal innervation was calculated as the percentage of area positive for β-tubulin staining as previously described.25 (link),26 (link)
4
FISH Assay for RNA Localization
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A fluorescence in situ hybridization (FISH) kit (RiboBio) was used in this work according to the manufacturer's protocol. In short, cells were fixed in 4% paraformaldehyde solution for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 5 minutes at 4°C. Then, we rinsed the cells with PBS. The RNA was hybridized with a Cy3-labeled probe for 18S, U6 and Rik overnight at 37°C and protected from light. Cells were rinsed with SSC (0.3 M NaCl and 0.03 M Na3 citrate) at 42°C, and the nuclei were then stained with DAPI. Fluorescence images were acquired with an LSM880 Zeiss inverted microscope (CarlZeiss, Germany).
5
Imaging Fibrin Clot Formation Kinetics
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Plasma was diluted to a final ratio of 1 in 6 with TBS and spiked with Alexa Fluor 594 labeled fibrinogen at 50 µg/mL final concentration. Clotting was triggered by the addition of 5 mM CaCl2 (final concentration) and either TF (final concentration, 1 pM) or thrombin (final concentration, 0.1 U/mL). Once clotting was initiated, the mixture was immediately transferred to the channel of an uncoated µ‐Slide VI 0.4 mm (Ibidi GmbH, Gräfelfing, Germany), and placed in a dark humidity chamber for 2 hours at room temperature to allow clots to form. Images were obtained using a Zeiss LSM880 inverted microscope with a 40× oil immersion lens. Optical z‐stacks (43 × 0.7 µm) were combined and flattened to show maximum intensity. Fiber density was determined by averaging the total number of fibers crossing an arbitrary straight line of fixed length (200 µm). Image processing and fiber counting were executed on ImageJ software. Fibrin clots were prepared in triplicate, and three density measurements were obtained per clot.
6
Confocal Imaging of C. elegans Males
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Day 1 adult males isolated from hermaphrodites as L4 larvae were anaesthetized with 10 mM levamisole and mounted on 10% agarose pads for imaging at room temperature. Confocal imaging was performed with a Zeiss LSM 880 inverted microscope with an Airyscan superresolution module using a LSM T-PMT detector and ZenBlack software (Carl Zeiss Microscopy, Oberkochen, Germany). Images were acquired using a 63x/1.4 Oil Plan-Apochromat objective in Airyscan Fast mode and deconvolved using Airyscan processing. Image files were imported into Fiji/ImageJ [99 ] with the BioFormats Importer plugin processing and analysis. Images were placed in Adobe Illustrator for figure assembly.
7
Immunostaining Protocol for Hepatitis C Virus Proteins and Lipid Droplets
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Cells were washed with PBS before fixation for 20 min in 4 % (w/v) PFA, and they were subsequently permeabilized in 0.1 % (v/v) Triton X-100, PBS and blocked with PBS-T, 5 % (w/v) BSA before immunostaining with the denoted antibody. The primary antibodies used were: anti-NS5A (sheep), 1 : 1,000; anti-NS3 (mouse), 1 : 350; and anti-PI4P (mouse), 1 : 100 (Echelon). Various fluorescently conjugated secondary antibodies were used at 1 : 1000 (Life Technology). LDs were stained using the BODIPY (558/568)-C12 dye at 1 : 5000 (Life Technology), added at the same time as the fluorescent secondary antibody. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Confocal microscopy images were acquired on a Carl Zeiss LSM 880 inverted microscope, and post-acquisition analysis was conducted using Zen software (Zen version 2012 black edition 8.0, Zeiss, Germany).
8
Imaging of Levamisole-Anesthetized Adult Males
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Day 1 adult males isolated from hermaphrodites as L4 larvae were anaesthetized with 10 mM levamisole and mounted on 4% agarose pads for imaging at room temperature. Confocal time lapse imaging was performed with a Zeiss LSM 880 inverted microscope with an Airyscan superresolution module using a LSM T-PMT detector and ZenBlack software (Carl Zeiss Microscopy, Oberkochen, Germany). Images were acquired using a 63x/1.4 Oil Plan-Apochromat objective in Airyscan Fast mode and deconvolved using Airyscan processing. Kymograph Clear and Kymograph Direct were used to generate and analyze kymographs [101 (link), 102 ].
9
Adenoviral Transduction of Islets for RcaMP1h Calcium Imaging
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The genetically encoded calcium sensor pRSET-RcaMP1h was a gift from Loren Looger (Addgene plasmid # 42874). The coding sequence of the RcaMP1h sensor was subcloned into the pShuttle vector, and recombinant adenovirus particles were produced following the AdEasy XL Adenoviral Vector System protocol (#240010 Agilent Technologies). Islets were transduced using a microfluidic device to obtain uniform infection of the islet cells throughout the islet volume and were incubated overnight before imaging. Imaging on islets was performed on the LSM880 inverted microscope (Zeiss Inc), using a heated stage-top incubator (Pecon GmbH) 24 hours post transduction. Islets were imaged in KRBH medium (NaCl 128.8 mM, KCl 4.8 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, CaCl2 2.5 mM, NaHCO3 5mM, HEPES 10 mM, pH 7.40, 0.1% BSA) supplemented with the desired glucose concentration. RcaMP1h fluorescence was excited with a 561 nm laser, and the emission was collected through a 570–650 nm bandpass.
10
Mitochondrial Membrane Potential Imaging
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For mitochondria staining, cells were incubated with a mitochondrial transmembrane potential indicator (TMRM; 125 ng/mL, Sigma) and MitoTracker green (5 mmol/L; both from Thermo Fisher Scientific) in complete culture media for 20 min at 37°C and assessed in confocal microscopy images. Microscopy was performed in an LSM 880 inverted microscope (Zeiss) using a 488 Argon Laser and a 543 nm solid state laser and a 63x oil immersion objective (N/A 1.4).
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