Brain sections were stained with FJB to detect degenerating neurons as described in our previous studies [27 (link);28 ;33 (link)]. Briefly, brain sections fixed with 4% PFA on glass slides were washed with ddH2O and immersed in 0.06% potassium permanganate for 20 min, and then immersed into 0.0004% FJB in 0.1% acetic acid solution for 45 min in the dark. For detecting apoptotic neurons, TUNEL staining was performed using an in situ cell death detection kit (Roche, CA) similar to our previous studies [18 ;36 ]. Briefly, brain tissue sections were incubated in freshly prepared permeabilization solution (0.1% triton x-100, 0.1% sodium citrate) for 2 min on ice. TUNEL reaction mixture was then added on the brain sections and incubated in a humidified atmosphere at 37°C for 60 min in the dark. Images of the FJB- and TUNEL-stained sections were acquired with MetaVue imaging software (Molecular Device, CA) using a Nikon epi-fluorescence microscopy (Nikon, Melville, NY) equipped with a CoolSNAP-EZ CCD-camera (Photometrics, Tucson, AZ).
Metavue imaging software
Manufactured by Molecular Devices
Sourced in United States
MetaVue is an imaging software solution that enables capture and analysis of images from various microscopy techniques. It provides a user-friendly interface for controlling imaging hardware and managing image acquisition workflows.
Automatically generated - may contain errors
Lab products found in correlation
Vectorshield containing dapi, by Vector Laboratories (2 mentions)
Eclipse te2000, by Nikon (2 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Ab14041, by Abcam (1 mentions)
Ab23750, by Abcam (1 mentions)
Oct embedding matrix, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Oris cell seeding stoppers, by Platypus Technologies (1 mentions)
Axio observer a1 microscope, by Zeiss (1 mentions)
Te2000 e, by Nikon (1 mentions)
Proteostat aggresome detection kit, by Enzo Life Sciences (1 mentions)
Neutral red, by Merck Group (1 mentions)
Eriochrome cyanine r, by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Fecl3 6h2o, by Merck Group (1 mentions)
Rhodamine solution, by Merck Group (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Alexa fluor 594 conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Atto 488 conjugated phalloidin, by Merck Group (1 mentions)
14 protocols using metavue imaging software
1
Detecting Neuronal Degeneration and Apoptosis
2
Embryonic and Postnatal Tendon Development
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From WT and Scx-Cre::Mmp14 lox/lox mice, whole hind limbs from E15.5 embryos, lower hind limbs without the skin from P0 pups and dissected Achilles tendons from P10 pups were cryo-preserved in OCT-embedding matrix (Thermo Scientific). Longitudinal sections of 8-µm thickness were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min and then blocked with 2% BSA in PBS for 1 hr. FN was detected using a rabbit polyclonal antibody (ab23750; Abcam; diluted 1:500), and periostin was detected using a goat polyclonal antibody (ab14041; Abcam; diluted 1:500). Cy3-conjugated secondary antibodies (Invitrogen) were used and sections were mounted using Vector Shield containing DAPI (Vector Laboratories). Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software (Molecular Devices).
3
Cell Migration Assay with Mitomycin
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Oris™ Cell Seeding Stoppers (Platypus Technologies, Madison, WI) were inserted into the wells of a 96 well dish to create the migration zone. Cells were then seeded at 9×103 cells per well and incubated for 18 hours to allow cell adherence. Stoppers were then removed and Mitomycin was added to the growth medium at a concentration of 1 µg/ml to inhibit cell proliferation. Cells were permitted to migrate into the wound area for 32 hours and were fixed and stained with 20% methanol, 1% crystal violet. Micrographs of the wound area were recorded with a Zeiss Axio Observer.A1 microscope and the size of the wound area was measured using the MetaVue imaging software (Molecular Devices).
4
TGF-β1 Induced Fibronectin Visualization
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Cells were grown on glass coverslips (22 × 22 mm) and treated with 2 ng/ml TGF-β1 for 24 h. The cells were fixed with 4% PFA and permeabilized with 0.05% Triton X-100 and then blocked with 3% milk in PBS for 30 min at room temperature. The cells were incubated for 1 h with antibodies to fibronectin (1:400) in 1% milk/PBS followed by incubation for 30 min with Texas red–conjugated secondary antibody (1:500) at room temperature. Fluorescence images were taken with a Plan Apochromat 60×/1.40 NA oil objective lens at ambient temperature using an inverted microscope (TE2000-E; Nikon) equipped with a charge-coupled device camera (CoolSNAP HQ; Photometrics). The images were acquired using MetaVue imaging software (v7.7.3, Molecular Devices).
5
Detecting Protein Aggregation in PHT Cells
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PHT cells were plated on glass coverslips and grown in TM medium (ScienCell Research Laboratories, Carlsbad, CA, USA). After several washes, the cells were incubated in serum- and growth-factor-free TM medium and then treated with vehicle/chloroquine (50 μM, Sigma-Aldrich, St. Louis, MO, USA) or normoxia/hypoxia–reoxygenation. For detection of aggregated protein, fixed cells were stained with ProteoStat dye using ProteoStat Aggresome Detection Kit (ENZO, ENZ-51023-KP002, Farmingdale, NY, USA) according to the manufacturer’s instruction. Then, coverslips were mounted in VECTASHIELD anti-fade mounting medium containing DAPI (VECTOR LABORATORIE, H-1200, Burlingame, CA, USA). Fluorescence images were captured using a Nikon Eclipse TE2000 (Nikon, Tokyo, Japan) fluorescent microscope and analyzed using MetaVue Imaging software (Molecular Devices, San Jose, CA, USA).
6
Quantifying Spinal Cord White Matter Sparing
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Spinal cord cryosections were prepared in the same manner as the sections used for immunohistochemistry. After being air-dried at room temperature and fixed by 4% paraformaldehyde in PBS for 10 min, the sections were incubated with 0.16% eriochrome cyanine-R (Sigma-Aldrich, St. Louis, MO, USA) and 10% FeCl3·6H2O (Sigma-Aldrich) in 0.5% H2SO4 (Sigma-Aldrich) for 30 min at room temperature. The sections were then washed with running tap water and differentiated in 1% ammonium hydroxide followed by counterstained with neutral red (Sigma-Aldrich). The areas of spared white matter were calculated by subtracting the cavity areas and gray matter from the total spinal cord sectional areas using MetaVue Imaging software (Molecular Devices Corporation). The portion of spared white matter was calculated as follows: (spared white matter/total area of spinal cord) × 100.
7
Microscopy Imaging of Fibrillarin Localization
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Microscopy experiments were done as described in69 . Briefly, cells were grown on glass coverslips (22 × 22 mm) and treated with inhibitors for 24 hours, then fixed with 4% PFA and permeabilized with 0.05% Triton X-100. For fibrillarin staining samples were blocked with 3% milk in PBS for 30 min at room temperature. The cells were incubated for 1 h with antibodies to fibrillarin (1:400) in 1% milk/PBS followed by incubation for 30 min with Texas red–conjugated secondary antibody (1:500) at room temperature. Fluorescence images were taken with a Plan Apochromat 60×/1.40 NA oil objective at ambient temperature using Nikon TE2000-E inverted microscope equipped with a charge-coupled device camera (CoolSNAP HQ; Photometrics). The images were acquired using MetaVue imaging software (v7.7.3, Molecular Devices).
8
Intravital Microscopy of Leukocyte Dynamics
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Intravital microscopy was performed using a Nikon Eclipse TE300 inverted microscope interfaced with a high-speed, high-sensitivity CCD camera Rolera em-c2 (QImaging, Canada). Briefly, mice were anesthetized using ketamine/xylazine (1/10 ratio), whole blood cells were labeled after an intravenous retro-orbital injection of 50 μL of 1 mg/mL rhodamine solution (Sigma) and the mesentery was externalized. Five minutes after the injection, a mesenteric venule of about 150 μm diameter was placed on the microscope (×20 objective) and the acquisition was recorded for 5 minutes using the MetaVue Imaging software (Molecular Devices, Sunnyvale CA). For each mouse, between 3 and 5 movies were recorded. Analyses were performed using Imaris software (Bitplane, Switzerland) using a standardized process and parameters were applied for all mice. The speed of each event (object detected using Imaris software) was automatically calculated. For each movie we obtained a mean of 10,000 events associated with a speed between 0 and 1000 μm/s. GraphPad Prism software (La Jolla, CA) was used to calculate event frequency distribution according to the speed, and results were normalized to the number of events for each movie. Results presented are the means of 3 movies per mouse.
9
Immunofluorescence Staining of V5-tagged Cells
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Cells fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS for 20 minutes at RT, then washed with PBS containing 0.1% (v/v) Tween 20 three times and blocked with 1% (w/v) bovine serum albumin, 0.1% (v/v) Triton X-100 in PBS containing 0.1% Tween 20 for 1 hour. Cells were incubated with primary antibodies, mouse monoclonal anti-V5 epitope or the appropriate control IgGs, diluted in the blocking buffer overnight at 4°C. Cells were washed with PBS containing 0.1% (v/v) Tween 20 for 10 minutes three times and incubated with Alexa Fluor 594-conjugated antibodies (Invitrogen) and Atto 488-conjugated phalloidin (Sigma) diluted with the blocking buffer for 1 hour at room temperature, protected from light. Stained cells were washed with PBS containing 0.1% (v/v) Tween 20 for 10 minutes three times and mounted using Vector Shield containing DAPI (Vector Laboratories). Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software (Molecular Devices).
10
Quantitative Autophagosome Morphometry
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Cells were cultured in SDCA medium to midlog phase, and autophagy was induced when necessary. Cells were harvested and subjected to fluorescence microscopy on an IX83 inverted system microscope (Olympus) equipped with a UPlanSApo100×/1.40 Oil (Olympus) and a CoolSNAP HQ CCD camera (Nippon Roper). A U-FGFP and U-FRFP filter sets (Olympus) were used for visualization of GFP/mNeonGreen and mRFP/FM 4-64, respectively. Images were acquired using the MetaVue imaging software (Molecular Devices). For determination of AM length, the fluorescence images of mNeonGreen-Atg8 were analyzed using Qautas (Kawaoka et al., 2017 (link)). FM 4-64 staining was performed as previously described (Suzuki et al., 2002 (link)). Data analysis was performed using R (https://www.r-project.org ). Boxplots were drawn using the default settings of R.
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