Cells were seeded and grown overnight on 12 mm circular glass slides in conditioned medium in a sterile 24-well tissue culture plate for 24 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min or cold methanol (20 min). Following fixation, cells were washed with 1x phosphate-buffered saline pH 7.4 (PBS) and stained with indicated lectins. SNA (biotinylated elderberry bark lectin, B-1305, Vector Laboratories) and MAL II (biotinylated Maackia amurensis lectin II, B-1265, Vector Laboratories) at concentrations of 10 μg/mL in PBS were incubated on slides for 1 hr at room temperature. Stained slides were then washed twice with PBS and incubated 1 hr with avidin-fluorescein (10 μg/mL). Cells without biotinylated lectins were used as controls. After three washings with PBS, slides were mounted in cell mounting media and analyzed with Carl Zeiss Imager 2 fluorescence microscope.
Imager 2 fluorescence microscope
Manufactured by Zeiss
The Imager 2 is a fluorescence microscope designed for high-resolution imaging. It features an advanced optical system and sensitive detectors to capture detailed images of fluorescently labeled samples. The Imager 2 is capable of capturing multi-channel fluorescence data, enabling the visualization of multiple targets within a single specimen.
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6 protocols using imager 2 fluorescence microscope
1
Lectin Staining of Cultured Cells
2
Evaluating Curcumin Loaded Nanoparticles in Multicellular Tumor Spheroids
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It is noteworthy that PANC-1 cells were unable to adhere to glass coverslips and thus generating spheroids for fluorescence microscopy was not possible, and were instead conducted with MDAMB231 cells. MDAMB231 cells were plated at 35,000 cells per glass coverslip on a 24-well plate. After 3 h, the cells were treated with 50 μM cyclo-RGDfK(TPP) peptide to facilitate spheroid formation as previously described elsewhere [10 (link)]. Changes in cellular aggregation and spheroid formation were observed using inverted phase-contrast microscopy. Following complete formation of MCTS, curcumin loaded SMA and FA-DABA-SMA were added directly to wells at a concentration 3 μM. Following 0.5 h and 6 h of treatment times, coverslips were removed from the wells and mounted on slides using DAPI containing fluorescent mounting media. MCTS were analyzed with Carl Zeiss Imager 2 fluorescence microscope at 100× and 200× magnification.
3
Lectin-based Glycoprofiling of Prostate Cancer Cells
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Cells were cultured in 24-well tissue culture plate with glass coverslips for 24 hours in CO2-incubator at 37°C. The PC3, DU145, and the chemoresistant variant cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature followed by washing with 1× PBS pH 7.4. Cells were incubated with MAL-II (biotinylated M. amurensis lectin II, B-1265; Vector Laboratories Inc.) and SNA (biotinylated elderberry bark lectin, B-1305; Vector Laboratories Inc.) at a concentration of 10 μg/mL in PBS for 1 hour at room temperature. The cells were washed three times with 1× PBS. After washing, the cells were incubated with streptavidin-conjugated Alexa Fluor 594 for 1 hour at room temperature in the dark. The background controls were cells with no biotinylated lectins added in the above procedure. After three washings with 1× PBS, slides were mounted on fluorescent mounting media and analyzed with Carl Zeiss Imager 2 fluorescence microscope at 200× and 400× magnification.
4
Fluorescence Microscopy of Nematodes
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The nematodes were placed in a 15 µl S-Basal plus levamisole 10 mM drop on a microscope glass slide, covered with a cover slide, and immediately imaged. Pictures were acquired with an Imager 2 Zeiss fluorescence microscope, with magnification 25-fold. The images were analyzed with the software ImageJ (http://imagej.nih.gov/ij/ ). The bright field images were used to generate the mask to select each single worm pictured, and consequently to measure the fluorescence in the correspondent fluorescent filter.
5
Quantifying hsp-6::GFP Expression in Nematodes
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For quantification of hsp-6::GFP expression, animals, exposed or not to 100 µg/ml cisplatin from L4/YA stage for 24 h, were transferred to a microscope slide and anesthetized with 15 µl of 10 mM levamisol hydrochloride (Sigma-Aldrich, 31742) dissolved in S-Basal [5.85 g NaCl, 1 g K2HPO4, 6 g KH2PO4, 1 ml cholesterol (5 mg/ml in ethanol), H2O to 1 l], and sealed with a cover slip. Worms were pictured at 25× magnification using an Imager2 Zeiss fluorescence microscope, and the same exposure time was applied to all experimental conditions. GFP expression was quantified using ImageJ software (https://imagej.nih.gov/ij/ ), measuring the fluorescence intensity of the pharyngeal bulb region of each worm acquired. Three experimental replicates were performed, and, for each condition, 10-15 worms were pictured and quantified.
6
Transgenic Nematode Phenotyping Assay
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The effect of nuo-5 and lpd-5 RNAi and/or lutein (Sigma Aldrich PHR1699) treatment on the induction of different transgenic strains was investigated on synchronized population of L3 worms (unless otherwise indicated). The nematodes were placed in a 14 μl S-Basal drop on a microscope glass slide, anesthetized with NaN3 10 mM, covered with a cover slide and immediately imaged. Pictures were acquired with an Imager2 Zeiss fluorescence microscope, magnification tenfold and ZEN 2 (blue edition) software. In each experiment a minimum of 15–20 animals per condition were used. Details for each fluorescent strain are described in Supplementary Methods.
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