Super-resolution confocal microscopy was performed to visualize the biofilm components (bacteria, fungi, and EPS) that become entrenched among the extended STARS bristles. The EPS glucan matrix was labeled with Alexa Fluor 647 dextran conjugate (Molecular Probes). S. mutans and C. albicans (if applicable) were stained with SYTO9 (Molecular Probes) and Concanavalin A-tetramethylrhodamine conjugate (Molecular Probes), respectively, as described in ref 44 (link). After biofilm removal, STARS bristles were collected and immobilized in 1% agarose for super-resolution imaging using a 40× water immersion objective (numerical aperture = 1.2) on an upright confocal microscope (Carl Zeiss LSM 800, Germany) with Airyscan. The STARS bristles were visualized using the reflection mode and a 405 nm laser. In a separate experiment, biofilm components entrenched in STARS bristles were dehydrated through a graded ethanol series and examined by SEM (FEI Quanta 600, FEI, Portland, OR, USA).
Concanavalin a tetramethyl rhodamine conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Concanavalin A Tetramethyl rhodamine conjugate is a fluorescently labeled lectin that binds to carbohydrates. It is used as a tool in cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (4 mentions)
Ez c1, by Nikon (3 mentions)
Alexa fluor 647 dextran conjugate, by Thermo Fisher Scientific (2 mentions)
Quanta 600, by Thermo Fisher Scientific (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Syto9, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Imaris, by Oxford Instruments (1 mentions)
Ab92824, by Abcam (1 mentions)
Dylight 488 goat anti mouse igg h l, by Abcam (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
7 protocols using concanavalin a tetramethyl rhodamine conjugate
1
Visualizing Biofilm Structures in STARS
2
Visualizing Biofilm Components in STARS
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Super-resolution
confocal microscopy was performed to visualize the biofilm components
(bacteria, fungi, and EPS) that become entrenched among the extended
STARS bristles. The EPS glucan matrix was labeled with Alexa Fluor
647 dextran conjugate (Molecular Probes). S. mutans and C. albicans (if applicable) were stained with
SYTO9 (Molecular Probes) and Concanavalin A-tetramethylrhodamine conjugate
(Molecular Probes), respectively, as described in ref (44 (link)). After biofilm removal,
STARS bristles were collected and immobilized in 1% agarose for super-resolution
imaging using a 40× water immersion objective (numerical aperture
= 1.2) on an upright confocal microscope (Carl Zeiss LSM 800, Germany)
with Airyscan. The STARS bristles were visualized using the reflection
mode and a 405 nm laser. In a separate experiment, biofilm components
entrenched in STARS bristles were dehydrated through a graded ethanol
series and examined by SEM (FEI Quanta 600, FEI, Portland, OR, USA).
confocal microscopy was performed to visualize the biofilm components
(bacteria, fungi, and EPS) that become entrenched among the extended
STARS bristles. The EPS glucan matrix was labeled with Alexa Fluor
647 dextran conjugate (Molecular Probes). S. mutans and C. albicans (if applicable) were stained with
SYTO9 (Molecular Probes) and Concanavalin A-tetramethylrhodamine conjugate
(Molecular Probes), respectively, as described in ref (44 (link)). After biofilm removal,
STARS bristles were collected and immobilized in 1% agarose for super-resolution
imaging using a 40× water immersion objective (numerical aperture
= 1.2) on an upright confocal microscope (Carl Zeiss LSM 800, Germany)
with Airyscan. The STARS bristles were visualized using the reflection
mode and a 405 nm laser. In a separate experiment, biofilm components
entrenched in STARS bristles were dehydrated through a graded ethanol
series and examined by SEM (FEI Quanta 600, FEI, Portland, OR, USA).
3
Visualizing Retinal Leukocyte Adhesion
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The rats (n = 5) were anesthetized with ketamine (100 mg / kg) and xylazine (8 mg / kg) cocktail, and an incision was made on the abdomen wall to access the chest cavity. Intracardiac perfusion was performed with 50 - 60 mL of pre-warmed saline (0.9% NaCl) for three min to remove the circulating red blood cells and leukocytes. This was followed by perfusion of 10 mL from 100 µg / mL of Concanavalin A Tetramethyl rhodamine conjugate (C 860, Invitrogen) for an additional two minutes. The eyes were enucleated and fixed in the 4% PFA for 2 h. The cornea and lens were separated, and the whole retina was extracted and mounted on the slide. Retinal images were taken using a fluorescent microscope (Olympus, Tokyo, Japan), and the number of adherent leukocytes was counted from the whole retina in each group.
4
Biofilm Exopolysaccharide Analysis with ILs
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Biofilm samples were treated with 10% hydrophilic ILs and left for 10 min at room temperature. Samples that were not treated with ILs were used as controls. The specimens were washed and then stained with Concanavalin A-tetramethylrhodamine conjugate (Invitrogen, Carlsbad, CA, USA) and SYTO® 9 (Invitrogen) for 30 min in the dark. After rinsing in water, images were obtained using a CLSM (LSM700, Carl Zeiss, Munchen-Hallbergmoos, Germany). The scanning images were analyzed three-dimensionally using imaging software (Imaris®, Bitplane AG, Zurich, Switzerland). Fluorescent images were also quantified and exopolysaccharide levels were calculated as the ratio of the numbers of exopolysaccharides and cells. Significant differences between experimental and control groups were analyzed using Dunnett’s multiple comparison test (P < 0.01). Eight images per field per sample were acquired randomly. The experiment was independently repeated three times.
5
Confocal Imaging of Fluorescent Liposomes in HepG2 Cells
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Imaging by confocal laser scanning microscope. The culture medium used to grow the HepG2 cells (HB-8065 ATCC) was DMEM supplemented with 10% FBS and 1% Pen-Strep and cells were seeded in 24-well plates at a density of 5×103 cells/well at 37°C in a humidified 5% CO2 atmosphere for 24 h. For the imaging studies, we followed the same procedure described in the literature (39 (link),40 (link)) where the medium was replaced with 0.3 mg/ml of Chit-Lip-FA solution in culture medium and incubated for 0.5, 4, 8 and 24 h; after incubation time with fluorescent liposomes, the cells were washed three times with PBS and fixed with 2.5% glutaraldehyde in PBS for 20 min. Concanavalin A tetramethylrhodamine conjugate (Invitrogen, Life Technology; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to stain the membrane at a final concentration of 100 µg/ml. After washing in PBS, human cells were blocked with 1% BSA in PBS for 20 min and washed three times with PBS. For data acquisition, we used a confocal microscope (C1 Nikon; Nikon Cor., Tokyo, Japan) with EZ-C1 software (Nikon Corp.) and 60× or 100× oil immersion objective, imaging of the fluorescent liposomes was evaluated by excitation/emission at 492/518 nm, and the cell membrane with excitation/emission at 555/580 nm.
6
Mitochondrial Changes in Human Cardiac Cells
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Morphological changes and mitochondrial activity of human cardiac cells were studied through a Confocal Laser Scanning Microscope (EZ-C1-Nikon). Briefly, human cardiac cells were untreated (control) or treated with DOXO alone or combined with DAPA for 24 h. After incubation, cardiomyocytes were fixed in 4% formaldehyde (10 min) and then incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween20 for 1 h to permeabilize the cells and block non-specific protein–protein interactions. The cardiomyocytes were then incubated with an anti-Mitochondria antibody (113-1)—BSA and Azide free (Abcam ab92824, Milan, Italy) 5 µg/ml overnight at ±4°C. As a secondary antibody (green), a DyLight® 488 goat anti-mouse IgG (H ± L) (ab96879, Abcam, Milan, Italy) was used at a dilution of 1/250 for 1 h. Membrane staining was obtained using Concanavalin A Tetramethylrhodamine Conjugate (Invitrogen, Life Technology, Milan, Italy) at a final concentration of 100 µg/ml. Through a confocal microscope (C1-Nikon) equipped with EZ-C1 software for data acquisition and 60× oil immersion objective, intracellular mitochondria were imaged through excitation/emission at 488/518 nm and cell membrane through excitation/emission at 555/580 nm (31 (link)).
7
Fluorescent Labeling of Biofilm Proteins
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