BAECs were directly plated on 24 mm2 glass cover slips and subsequently used for evaluating endogenous NO production. Cells, previously deprived of serum and treated with insulin, EAE, or MA, as previously described (see cell culture experiments), were incubated with a fluorescent NO indicator 4,5-diaminofluorescein diacetate (DAF-2DA, 10−5M; Sigma-Aldrich, USA) for 30 min. Thereafter, cells were washed with cold PBS and fixed in paraformaldehyde (PFA) at 4%. Nuclei were stained by incubation with 4′,6-diamino-2-phenylindole (DAPI, 1:500 from stock 5 mg/ml; Molecular Probes, USA) for 15 min at room temperature in the dark and cells were washed with PBS. The cover slips with BAECs were mounted in a glycerol/PBS solution (Citifluor, VWR International, Spain) and analyzed by confocal microscopy as previously described28 (link). From each culture, a minimum of 3 randomly selected images were captured with a LEICA SP5 confocal microscope (Leica Microsystems) using the 488 nm/530 nm (DAF-2DA, NO) and 405 nm/410–475 nm (DAPI, nuclei) filters with a x20 objective.
Citifluor
Manufactured by Avantor
Sourced in Spain, United Kingdom, Belgium
Citifluor is a glycerol-based anti-fade mounting medium designed to protect fluorescent signals in microscopy applications. It helps preserve the fluorescence of labeled samples during imaging.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
4 5 diaminofluorescein diacetate daf 2da, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Leica (1 mentions)
Axiophot, by Zeiss (1 mentions)
Axioskop fluorescent microscope, by Zeiss (1 mentions)
Rabbit anti collagen 4, by Abcam (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Ultraview vox, by PerkinElmer (1 mentions)
Saponin, by Carl Roth (1 mentions)
Eclipse ti e inverted, by Nikon (1 mentions)
4 protocols using citifluor
1
Quantifying Endogenous Nitric Oxide in BAECs
2
Midgut Morphology Examination of Drosophila Embryos
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Embryos aged 18–24 hours AEL were collected on apple-juice-agar plates from a cage held at 25°C. Embryos were dechorionated with commercial bleach for 3 min and rinsed with water. The embryos were transferred to a clean microscope slide and mounted in Citifluor (VWR) under a coverslip. The midgut morphology was examined using a standard Zeiss Axiophot microscope (Filter: BP 546; FT 580; LP 590).
3
Immunofluorescent Staining of Murine Retinal Flat Mounts
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Mice were culled by cervical dislocation, and eyes for flat mounts were removed with forceps and fixed in 4% paraformaldehyde (PFA) for 30 min, before storing in PBS. Eyes were dissected so that only RPE-choroid remained. To enable the eyecup to be spread flat, four radial cuts in the sclera-choroid-RPE were made. Eyecups were washed four times in PBS, before blocking with 15% normal goat serum IH buffer (PBS + 0.2% Tween + 0.5% BSA), with shaking on an orbital shaker at 100 rpm overnight at 4°C. Samples were then incubated with a 1:500 dilution of rabbit anti-collagen IV (Abcam) in IH buffer for 2 days shaking at 4°C. Samples were washed four times in PBS, before incubating with a 1:200 dilution of goat anti-rabbit Alexafluor594 (Invitrogen) in IHC buffer for 1 day at 4°C. Samples were washed three times in PBS, mounted on a Superfrost Plus slide (Fisher Scientific) in 0.25 mL pre-warmed mowiol+citifluor (16% Mowiol; Harlow Chemicals, Batley, UK) in 30% glycerol in PBS plus 0.1% citifluor (VWR, Lutterworth, UK), and sealed with a coverslip. Slides were incubated overnight at 4°C in the dark to dry. Images were taken using a Zeiss Axioskop fluorescent microscope.
4
Imaging Intracellular Lipid Accumulation
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In three replicates, monolayers from 3 inserts per treatment were fixed at Day 10 of culture (and after NEFA exposure according to Fig. 1) in 4% phosphate buffered paraformaldehyde for 10 min. BOECs were washed twice with DPBS and permeabilized with saponin (0.1% w/v) (Carl Roth GmbH&Co, Karlsruhe, Germany). Nuclei were stained with 5 µg/mL DAPI (Molecular Probes) for 5 min and subsequently washed with DPBS. Neutral lipids were stained with BODIPY 493/503 (Molecular Probes) (20 µg/mL) in DPBS for 1 h, according to a modified protocol of Van Hoeck and coworkers (Van Hoeck et al. 2013) . After staining the insert membranes and monolayers were removed from the insert housing and mounted on a microscope slides with Citifluor (VWR, Haasrode, Belgium). High-resolution images were obtained using Nikon Eclipse Ti-E inverted microscope, attached to a microlens-enhanced dual-spinning disk confocal system (UltraVIEW VoX; PerkinElmer) equipped with 405 and 488 nm diode lasers for the excitation of blue and green fluorophores respectively. For each monolayer, 10 random z-stack of 20 µm with each 1 µm intervals were made starting at the level of the insert membrane. In extended focus images, neutral lipid accumulation was compared qualitatively among treatments.
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