Transfected HEK293AD cells fixed (HOW) on 18 mm glass coverslips were placed in 12-well Corning plates (Corning, Durham, NC, USA; catalog #3513) and washed with PBS (Sigma-Aldrich; catalog # D8537), then incubated for 10 min at room temperature with PBS + 0.25% Triton X100 (Research Products International, Mt. Prospect, IL, USA; catalog # 111,036). Coverslips were blocked in normal goat serum (Vector Laboratories, Newark, CA, USA; catalog # S-1000) for 1 h at room temperature, then incubated with one of the 7TM primary antibodies (1:500) and FLAG antibody (1:500, Abcam, Cambridge, UK; catalog # 117,495) overnight at 4 °C. The following day, coverslips were washed 3X with PBS and incubated with AlexaFluor488 goat anti-rabbit antibody (1:1000, Invitrogen, Waltham, MA, USA; catalog # A11008) for 1 h at room temperature. Coverslips were washed 3X with PBS and mounted on glass slides with ProLong Gold with DAPI (Vector Laboratories; catalog # H-1500). Slides were dried and imaged on a Nikon Eclipse Ti2-E Inverted Motorized Research Microscope with Perfect Focus System 4 and NIS-Elements AR Software (AI algorithms for optimal signal/noise imaging; 2D/3D deconvolution). Images shown are at 40 × magnification with exposure time of 50 ms for fluorescein isothiocyanate (FITC) and 400 ms for tetramethylrhodamine (TRITC) fluorophore channels.
Prolong gold with dapi
Manufactured by Vector Laboratories
ProLong Gold with DAPI is a mounting medium for fluorescence microscopy. It is designed to preserve and protect fluorescent signals while providing nuclear counterstaining with DAPI.
Automatically generated - may contain errors
2 protocols using prolong gold with dapi
1
Immunofluorescence of Transfected HEK293AD Cells
2
Immunofluorescence analysis of ADI-PEG20 treatment
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5000 cells were seeded in 2-well chamber slides. Cells were treated with 1 μg/mL ADI-PEG20 (Polaris Inc, San Diego, CA, USA) for 72 h. Cells were washed twice with 1x PBS and fixed with 4% PFA in 1x PBS for 10 min. Cells were washed three times with 1x PBS and blocked with 50% goat serum in 1x PBS for 30 min at 37 ºC. Primary antibodies were then added for 1 h at 37 ºC, washing three times for 5 min. Secondary antibodies were added for 1 h at 37 ºC. After a final three washes for 5 min, cells were mounted in Prolong Gold with DAPI (Vector, Olean, NY). Cells were imaged with an Olympus microscope and an Olympus DP72 camera. Camera settings were 128 ms for all FITC images. All images were assembled with Photoshop (Elements 13) and all changes to the FITC images were uniformly applied for figure clarity Photographs were equally adjusted in the green channel for printing clarity by changes “levels” so that the maximal intensity of the control figure was the given the maximal grey value. This was applied equally to the entire dataset. The nuclear stain in blue was adjusted non-uniformly as it is a nuclear marker to be used as a cellular special reference and not part of the experiment.
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