Cells grown on cover slips were either treated with 1 mM DFMO or untreated. Cells were fixed with 4% formalin for 15 minutes, washed with PBS, permeabilized and blocked with 0.2% Triton X-100 and 2% BSA in PBS (blocking solution) for 30 minutes at room temperature (RT). Cells were sequentially incubated as follows: Primary rabbit anti-hACE2 (Invitrogen, SN0754) (Vero E6) or rabbit anti-mCEACAM (BHK-R) antibodies 1:500 in blocking for 2 hrs at room temperature. Cells were subsequently washed then incubated with secondary goat anti-rabbit (1:500 in blocking, 30 min, RT). Mounting media with DAPI was used to visualize nuclei. Samples were imaged with Zeiss Axio Observer 7 with Lumencor Spectra X LED light system and a Hamamatsu Flash 4 camera using appropriate filters using Zen Blue software with a 40X objective.
Spectra x led
Manufactured by Hamamatsu Photonics
The Spectra X LED is a compact and versatile light source designed for fluorescence microscopy and other scientific applications. It provides a broad spectrum of LED-based illumination, allowing users to select the desired wavelength. The Spectra X LED offers adjustable light intensity and can be controlled through a user-friendly software interface.
Automatically generated - may contain errors
Lab products found in correlation
Flash 4, by Hamamatsu Photonics (2 mentions)
Axio observer 7, by Lumencor (1 mentions)
Plan apo 1.4 na objective, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
Cmos camera, by Hamamatsu Photonics (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
3 protocols using spectra x led
1
Immunofluorescence Analysis of ACE2 and CEACAM
2
Assaying mRNA Export Defects using FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
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To assay for mRNA export defects, FISH against poly(A)-RNA was performed as previously described (Cole et al., 2002 (link)). In brief, REF (BMY83) and mex67-5 (BMY135) strains were grown to mid-log phase at a permissive temperature (26°C) and then shifted to a nonpermissive (37°C) temperature for 30 min with prewarmed media. After fixation, poly(A)-RNA was detected using a fluorescein-labeled dT50 probe, and DNA was visualized using DAPI. Imaging was performed on a microscope (DeltaVision Elite) equipped with a front-illuminated sCMOS camera driven by softWoRx 6 at 23°C using a 60× 1.4 NA oil objective. To localize Mex67p, haploid strains were generated (KWY5566 and KWY5567) expressing Ndc1p-tdTomato, GFA1-PP7, and GFP-tagged Mex67. To avoid cross talk from the PP7-CP tagged with YFP, we used strains that did not express the coat protein. Cells were grown in a synthetic complete medium at 26°C and then imaged in a 384-well plate coated with concanavalin A at 26°C using an inverted epifluorescence microscope (Ti; Nikon) equipped with a Spectra X LED light source and an sCMOS camera (Flash 4.0; Hamamatsu Photonics) using a 100× Plan-Apo 1.4 NA objective and the NIS Elements software (Nikon). All image processing was done using Fiji (Schindelin et al., 2012 (link)).
3
Heat Shock Stress Imaging
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