Cells were fixed in 4% paraformaldehyde in PBS for 15 min, exposed to 0.5% Triton X-100 with 10% goat serum in 1X PBS for 2 hours at room temperature while shaking. The fixed cells were then incubated overnight with a rabbit polyclonal antibody directed against ZO-1 (1:500 dilution, Thermo Fisher Scientific) at 4°C while shaking. Cells were then washed and a secondary antibody conjugated to AlexaFluor 555 (1:1000, dilution, Life Technologies, USA) was added for 1 hour at room temperature in the dark. After that cells were washed with 1X PBS and Alexa Fluor 488 Phalloidin (A12379, 1:40 dilution, Life Technologies, USA) was used for staining F-actin. To that end, cells were incubated for 20 minutes at room temperature with Alexa Fluor 488 Phalloidin at 1:40 dilution and then washed with 1X PBS. Slides were then counterstained with DAPI, washed with 1X PBS, mounted with a ProLong Gold anti-fade reagent (ProLong Gold, Life Technologies, USA), coverslipped, and examined using a Nikon Eclipse 80i fluorescence microscope (Nikon USA, Melville, NY). Imaging was performed using a CoolSNAP EZ CCD camera and NIS-elements software (Nikon USA).
Coolsnap ez ccd camera
Manufactured by Nikon
Sourced in United States
The CoolSNAP EZ CCD camera is a high-performance imaging device designed for scientific and industrial applications. It features a charge-coupled device (CCD) sensor that captures detailed images with high resolution and sensitivity. The camera is capable of capturing images with a range of exposure times and provides a digital output for further analysis and processing.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Eclipse 80i fluorescence microscope, by Nikon (1 mentions)
Nis element software, by Nikon (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Epifluorescence microscope, by Nikon (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Ixon 897 emccd camera, by Oxford Instruments (1 mentions)
Structured illumination microscope n sim, by Nikon (1 mentions)
Eclipse ti microscope system, by Nikon (1 mentions)
5 protocols using coolsnap ez ccd camera
1
Immunofluorescence Staining of Tight Junctions
2
Measuring Worm Dehydration in Osmotic Conditions
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Worms were cultured on regular NGM plates (∼150 mOsm). To measure the dehydration caused by different osmotic conditions, a worm was first transferred onto an agar plate that had the same osmolarity as the cultivating plate to take pictures with an Eclipse TE2000U microscope with a CoolSNAP EZ CCD camera (Nikon). The worm was then soaked in a 200 or 400 mOsm solution for 8 min and returned to the original agar plate to take another set of pictures. Three pictures were taken for each worm under each condition. The outline of each worm was manually traced, and the area of the worm body was measured with the NIS-element software (Nikon) in pixels. To minimize the subjective bias during the measurement, the picture files were randomly ordered and renamed using a MATLAB code. The measurement was performed with the researcher blind to the conditions.
3
Scalpel-Mediated Dye Transfer Protocol
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A scalpel loading-dye transfer (SL-DT) technique was adapted after the method of El-Fouly et al. [26 (link)]. After treatment with the chemicals, cells were washed with phosphate buffered saline containing 0.1 g/L calcium chloride and 0.1 g/L magnesium chloride (CaMgPBS) followed by the addition of 1 mg/ml of Lucifer-Yellow dissolved in CaMgPBS. The dye was introduced into the cells with three different lines of scalpel-based dye injection through the monolayer of confluent cells using a surgical steel scalpel blade. The transfer of dye through gap junctions was for three minutes, followed by a thorough rinse with CaMgPBS to remove extracellular dye, and then fixed with a 4% formalin solution in PBS. Migration of the dye in the cells was observed at 200X using a Nikon epifluorescence microscope equipped with a Nikon Cool Snap EZ CCD camera and the images digitally acquired using a Nikon NIS-Elements F2.2 imaging system. The fluorescence area of the dye migration from the scalpel line was quantified using ‘ImageJ’ image analysis program (National Institute of Health, Bethesda, MD). The data were reported as a fraction of the dye spread in the vehicle control (Fraction of the Control, FOC).
4
Differential Interference Contrast Imaging of Fibrils
5
Imaging Subcellular Localization of GalT
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Images of GalT were captured using a Nikon Eclipse Ti Microscope System equipped with NIS-Elements AR imaging software (v. 4.60). Images were taken using a Nikon 60× oil-immersion lens (Plan Apo VC, 1.4 NA) and a Photometrics CoolSnap EZ CCD camera and deconvolved using Nikon deconvolution software (v. 4.40). Superresolution microscopy was performed on a Nikon Structured-Illumination Microscope (N-SIM; Nikon) using NIS-Elements AR imaging and 3D reconstruction software (v. 5.11). SIM images were taken using a heated Nikon 100× oil-immersion lens (CFI Apo SR TIRF, 1.49 NA) and an Andor iXon+897 EMCCD camera. Live-cell images on the SIM were captured every 3 min, beginning 20 min after biotin addition. Confocal microscopy was performed on a Nikon Eclipse Ti2 microscope equipped with a W1 Spinning Disc, Orca Flash CMOS camera, and 60× oil-immersion objective (CFI Plan Apo λ, 1.4 NA), and NIS-Elements AR imaging and 3D reconstruction software (v. 6.0). All microscopes were equipped with motorized stages, Tokei Hit environmental chambers (temperature-controlled at 37°C and 5% CO2), and Nikon Perfect Focus motors. Live-cell images were captured every 90 s, beginning just after biotin addition. Video and still images were deconvoluted using Nikon Elements deconvolution software (v. 4.51, 5.11, or 6), exported as TIF files, and cropped using Adobe Photoshop CS5 (Adobe, San Jose, CA).
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