BMDMs (2 × 105) from transgenic p65-DsRedxp/IκBα-eGFP mice plated in 35 mm MatTek glass bottom microwells (MatTek Corp.; Ashland, MA, USA), in RPMI 1640 medium containing 10% v/v FBS. Cultures were stimulated with 100 ng/mL of LPS, and imaged for 5 h using a Leica LSM-800 confocal microscope (488 nm and 561 nm lasers)
Lsm 800 confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica LSM-800 is a confocal microscope designed for high-resolution imaging. It features a laser scanning system, which allows for the capture of detailed optical sections within a specimen. The LSM-800 is capable of producing three-dimensional reconstructions of samples by compiling these optical sections.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Axiocam hrc camera, by Zeiss (2 mentions)
Axio imager m1 microscope, by Zeiss (2 mentions)
Speinv inverted confocal microscope, by Leica (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Thunder widefield microscope, by Leica (1 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Donkey anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Goat anti gfp, by Abcam (1 mentions)
Anti ubiquitin, by Thermo Fisher Scientific (1 mentions)
Mouse on mouse polymer ihc kit, by Abcam (1 mentions)
Thunder imager, by Leica (1 mentions)
Fluorol yellow 088, by Santa Cruz Biotechnology (1 mentions)
Dextran alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Epifluorescence microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
15 protocols using lsm 800 confocal microscope
1
Imaging LPS-Induced NF-κB Dynamics
2
Immunofluorescence Imaging of HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stable HEK293T lentiviral cell-lines expressing RpH-LAMP1-3xFLAG were fixed in 4% PFA and 4% sucrose in PBS and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, X100) for 20 min. Samples were blocked in 0.1% BSA (Sigma-Aldrich, A7906) for 1 h at room temperature prior to incubation with primary antibody overnight at 4°C. Samples were then incubated with goat anti-mouse Alexa-Fluor-647 secondary antibodies for 1 h at room temperature. Samples were mounted using ProLong Gold Antifade reagent (Life Technologies, P36930) prior to imaging on Leica LSM-800 confocal microscope using 488 nm, 561 nm and 647 nm lasers and 63x objective. Nuclei were stained using DAPI (Sigma, D9542) (1:2000)
3
Organoid Segmentation Dataset Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the development of the OrgaSegment model we created a dataset containing images of organoids with various degrees of residual CFTR function. Organoids were cultured as described, and after 1 day imaged at different conditions (with or without forskolin and CFTR modulators) with a Zeiss LSM800 confocal microscope using 5x objective and transmitted laser light (ESID) or with a Leica Thunder widefield microscope using 5x objective and bright-field. Images were converted to JPEG, randomized to exclude any experiment information that could influence objective labelling, and uploaded to Labelbox24 for object labelling. Individual organoids were labelled into a single category (organoid). Labelling was performed by a group of experts, with experience in intestinal organoid culturing. Each image was labelled once and all labels were independently reviewed as quality control, and marked for re-labelling when of insufficient quality. The labelled dataset contained a total of 231 images (15,515 individual organoids), which were randomly split into training, validation and evaluation datasets, yielding datasets of 184 (11,989 organoids), 35 (2552 organoids), and 12 (974 organoids) grayscale images for respectively training, validation, and evaluation.
4
Immunostaining of Drosophila Nervous System
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Third instar larvae or adult fly brains were dissected in 1 X phosphate buffer saline (PBS) and fixed for 25 min in PBS containing 4% paraformaldehyde. Fixed samples were washed rapidly twice with PBS and three times for 15 min with PBS-0.3% Triton X-100 (PBT) before being pre-incubated for 1 h in PBT-1% bovine serum Albumin (BSA, Sigma). Samples were incubated overnight at 4 °C with primary antibody diluted in PBT-1% BSA, washed three times for 15 min in PBT, and incubated with respective secondary antibodies diluted in PBT-1% BSA for 3 hr at room temperature or overnight at 4 °C. Samples were washed in PBT and mounted in Vectashield-DAPI (Vector Laboratories). Images were acquired using a Leica LSM800 confocal microscope. The following antibodies were used: goat anti-GFP (Abcam, 1/500), mouse anti-Fasciclin II (DSHB, 1/25), donkey anti-goat Alexa Fluor 488 (Invitrogen, 1/1000), donkey anti-mouse Cy3 (Jackson Immuno, 1/1000).
5
Immunofluorescence Imaging of 3D Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence of 3D cultures, frozen sections were fixed with 1:1 ice-cold methanol:acetone. Blocking was performed using the Mouse on Mouse Polymer IHC Kit according to the manufacturer's protocols (ab127055; Abcam). Slides were incubated with anti-α-syn (syn211; 1:100 dilution; Invitrogen) and anti-ubiquitin (1:1000; cat. no. ab7780; Abcam) primary antibodies for 1 h at room temperature. Following PBS-T washes, secondary antibodies [Alexa Fluor® 488 goat anti-rabbit IgG (H+L) cat. no. ab150077, Abcam, and Texas Red™ goat anti-mouse IgG (H+L) cat. no. 100125662, ThermoFisher Scientific] were incubated at 1:1000 dilution for 1 h at room temperature. Nuclei were counterstained with 0.1 µg/ml DAPI (Sigma-Aldrich) for 5 min. Slides were mounted with immersion oil, sealed with nail varnish and left to cure overnight at 4°C in the dark before image capture using a Leica LSM 800 confocal microscope.
6
Quantifying UPEC Invasion of BECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human 5637 BECs were seeded on glass coverslips at approximately 1 × 105 cells/well in tissue culture-treated 24 well plates for a period of 16 h. Seeded cells were infected with different UPEC strains at MOIs ranging from 20–400 for a period of 1 h. After infecting the cells, gentamicin (200 μg/ml) and methyl D-mannose (0.5%) were added to the media and incubated for 30 min to remove the extracellular UPECs. D-mannose to media prevents UPEC from reattaching and entering the BECs. Prolonged incubation was performed in the media containing gentamicin (50 μg/ml) and methyl D-mannose (0.5%). The cells were fixed with 4% paraformaldehyde at relevant time points in the experiment for 20 min at RT. The fixed cells were permeabilized and blocked with 0.1% saponin in 1% BSA for 1 h. The blocked cells were treated with a primary antibody and followed by appropriate fluorophore-conjugated secondary antibodies. The examination of slides was performed using a Zeiss LSM800 confocal microscope or Lecia Thunder imager. The processing of images or quantification was determined using the image processing software Image J (National Institutes of Health).
7
Suberin Staining of Plant Leaves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S. viridis leaves were collected three weeks after germination and dehydrated in a series of washes with ethanol at increasing concentrations (70%, 90%, 100%) at room temperature (25°C) before being stored in 100% methanol at −20°C. Next, leaf cross sections were generated with a scalpel under a stereoscope. For suberin staining, the cross-sectioned samples were incubated in a freshly prepared solution of 0.01% (w/v) Fluorol Yellow 088 (Santa Cruz Biotechnologies) in lactic acid at 70°C for 1 hour2. The samples were washed two to three times with water and then incubated in Calcofluor White 0.1% (w/v) for 1 minute to stain the cell wall. Samples were gently inserted between agarose gel plates using fine forceps and transferred onto a glass slide, covered with a coverslip, and imaged on a Leica LSM800™ confocal microscope. Fluorol Yellow signal was detected using 488 nm excitation and 540–570 nm collection wavelengths. Cell membrane autofluorescence was detected using 405 nm excitation and 410–450 nm wavelengths. The brightness and contrast were adjusted by FIJI software.
8
Modulation of Lysosomal Dynamics in HEK293T and Cos7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stable HEK293T and lipofectamine transfected Cos7 lentiviral cell-lines expressing RpH-LAMP1-3xFLAG were seeded onto glass-bottom imaging dishes at the desired density for each experiment. 24 h post-seeding, cells were treated under one of the following conditions: 90 mM sucrose 4 h; 20 nM apilimod 3 h; 100 μM chloroquine 5 h; 250 nM torin2 2 h; 100 nM bafilomycin A1 plus 10 μM nigericin 30 min. Following treatment, cells were immediately imaged at room temperature in imaging buffer (Life Technologies) on a Leica LSM-800 confocal microscope using 488 nm and 561 nm lasers and 63x oil objective.
9
Lysosome Tracking in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stable HEK293T lentiviral cell-line expressing RpH-LAMP1-3xFLAG and untransduced HEK293T cells were seeded as a co-culture onto glass-bottom imaging dishes at the desired density for each experiment. 24 h post-seeding, cells were loaded with 20 μg/ml Alexa Fluor 647-Dextran (Thermo Fisher Scientific, D22914) for 16 h to label all lysosomes at 37°C 5% CO2, and chased for 3 h the following day at 37°C 5% CO2 prior to live-cell imaging on a Leica LSM-800 confocal microscope using 488 nm, 561 nm and 647 nm lasers and 63x oil objective.
10
Isolation and Imaging of Lysosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intact lysosomes were isolated from HEK293 T cells stably expressing lentiviral RpH-LAMP1-3xFLAG using anti-FLAG magnetic beads as described above. Lysosome-bound anti-FLAG magnetic beads were imaged on a Leica LSM-800 confocal microscope using 488 nm and 561 nm lasers and 63x oil objective. Z-stack images were collected at room temperature in fractionation buffer at intervals specified in the figure legends.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!