The 293T cells were inoculated in a gelatin‐coated 24‐well plate. After cotransfection with pmCherry‐IRF7 and EGFP‐myc‐ATF3 for 48 hours, the cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X‐100 and incubated in Image‐IT™ FX signal enhancer (I36933, Invitrogen) for 30 minutes. Following washing with TBS‐T and DAPI staining, the slides were mounted using mounting solution (D2522; Sigma). Images were obtained with a confocal laser‐scanning microscope (Fluo View 1,000; Olympus).
D2522
Manufactured by Merck Group
The D2522 is a laboratory equipment product manufactured by Merck Group. It is designed for general scientific and research applications. The core function of the D2522 is to provide a reliable and precise measurement tool for various laboratory tasks. However, a detailed and unbiased description of the product's features and intended use cannot be provided without the risk of making interpretations or extrapolations beyond the scope of a factual presentation.
Automatically generated - may contain errors
Lab products found in correlation
Image it fx signal enhancer, by Thermo Fisher Scientific (3 mentions)
Fluoview 1000, by Olympus (3 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
D9564, by Merck Group (1 mentions)
D8418, by Merck Group (1 mentions)
Axio imager m1 compound microscope, by Zeiss (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Image j software, by Adobe (1 mentions)
Alexa fluor 488 anti rabbit, by Jackson ImmunoResearch (1 mentions)
Ab21600, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Ht25a 1kt, by Merck Group (1 mentions)
8 protocols using d2522
1
Subcellular Localization of IRF7 and ATF3
2
Visualizing ASK1-TRAF1 Interactions
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HepG2 cells were seeded onto coverslips in 24-well plates. After co-transfection of pCherry-ASK1 and pEGFP-TRAF1 for 48 h, the cells were fixed with 4% fresh paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with 1 g/ml DAPI for 15 min. Between each step, the coverslips were washed three times with 1 × PBS. The coverslips were removed from the wells and placed on a slide with mounting solution (D2522, Sigma). Images were acquired with an Olympus FV1000 confocal microscope.
3
Immunofluorescence Imaging of Protein Interactions
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After co-transfection with pEGFP-SIKE and pmCherry-TBK1, the HEK293T cells were cultured on gelatin-coated coverslips in 24-well plates for 48 h. The cells were fixed with 4% fresh paraformaldehyde for 15 min, followed by three washes with PBS, 5 min of permeabilization with 0.2% Triton X-100 in PBS and subsequent incubation in an Image-IT FX signal enhancer (I36933, Invitrogen) for 30 min. The cells were then washed three times with TBST and stained with DAPI (1 g ml−1, 15 min). The slides were mounted with mounting solution (D2522, Sigma), and images were acquired using a confocal laser-scanning microscope (Fluoview 1000; Olympus).
4
Immunostaining of Pectoral Fins
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Pectoral fins were fixed overnight (o/n) at 4°C in a solution of 20% DMSO (Sigma, #D8418) in Methanol (MetOH). The following day, fins were rehydrated in MetOH/PBST0.3 (0.3% Triton X-100 in PBS-phosphate buffered saline) series, and then permeabilized with acetone for 20 minutes (min) at −20°C. After several washes with PBST0.3, fins were included for at least 1 hour in blocking solution (1% BSA, 1% goat serum, 1% DMSO in PBST0.3) at room temperature (R.T.). Incubation with primary antibody (ab.), diluted in blocking solution, took place o/n at 4°C. Non-conjugated antibody was removed by several washes with PBST0.3 and appropriate secondary alexa fluor ab. (Molecular Probes) was diluted in blocking solution, and left o/n at 4°C in the dark. Several washes with PBST0.3 were performed to remove the excess ab., and samples were counterstained with 0.15% (w/v) DAPI (4',6-diamidino-2-phenylindole, Sigma #D9564) in PBS, for 30 min. At the end, fins were washed in PBS and stored in mounting medium (2% DABCO, Sigma #D2522 and 80% Glycerol in PBS) for microscope analysis.
5
Immunofluorescence Imaging of Transfected H9C2 Cells
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H9C2 cells were grown on coverslips in 24-well plates. After co-transfection with pmCherry-IRF8 and pEGFP-NFATc1 for 48 h, the cells were fixed using 4% fresh paraformaldehyde for 15 min, followed by three washes with PBS and 5 min of permeabilization with 0.2% Triton X-100 in PBS, followed by incubation in an Image-IT FX signal enhancer (I36933, Invitrogen) for 30 min. Subsequently, the cells were washed three times with TBST and were stained with DAPI (1 g ml−1, 15 min). The slides were mounted with mounting solution (D2522, Sigma), and images were acquired using a confocal laser-scanning microscope (Fluoview 1000; Olympus).
6
Imaging Techniques for Embryo Analysis
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Embryos 24 h and older were deyolked in 70% glycerol/30% sterile water using mounting pins. For lateral and dorsal views of the embryo, whole embryos were mounted in 70% glycerol in coverslip sandwiches (24 × 60 mm coverslips; VWR, 48393-106), with 2–4 coverslips (22 × 22 mm; VWR, 16004-094) on either side of the sample to avoid sample compression. For ventral views of putative taste receptors, the trunk was dissected with a razor blade and the head carefully inverted on to a 24 × 60 mm coverslip and a similar coverslip sandwich made. For lateral views of eyes, they were dissected from forebrain using mounting pins and mounted as for whole embryos, but using only 1–2 coverslips each side of the specimen. Cross-sections were cut by hand using a razor blade mounted in a 12 cm blade holder (World Precision Instruments, Cat. #14134). Differential interference contrast (DIC) pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. A Zeiss LSM 710 confocal microscope was used to image embryos mounted in DABCO (1,4-Diazabicyclo[2.2.2]octane, Sigma, D-2522, 2% w/v solution in 80% sterile glycerol) for fluorescent double-labeling experiments. Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abràmoff et al., 2004 ).
7
Immunofluorescent LC3 Staining Protocol
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For LC3 staining, samples were fixed in ice‐cold methanol for 5 min at room temperature, followed by rehydration in PBS and processing for immunofluorescence. Serum starvation in 0.5% FBS was included as a control for induction of LC3 puncta. Fixed samples were rinsed 3–5 times in blocking buffer (2% BSA, 0.1% Triton X‐100, 0.1% NaN3 in PBS). Samples were incubated with LC3 diluted in blocking buffer for 1 h at room temperature. Samples were rinsed 3–5 times in blocking buffer for 5–10 min each before incubating in secondary antibodies (1 : 500) for 45 min at room temperature. Secondary antibodies and phalloidin were diluted in blocking buffer. Samples were rinsed 3–5 times in PBS, 5–10 min each, and then mounted in Mowiol mounting medium (0.1 M Tris/HCl, pH 8.5, 25% glycerol, 10% Mowiol 4‐88; 475904, Calbiochem, 2% DABCO; D2522, Sigma). All secondary fluorescently labelled antibodies were highly cross‐absorbed secondary antibodies from Jackson ImmunoResearch, Alexa Fluor® 488 Anti‐Rabbit (711‐545‐152) and Rhodamine (TRITC) Anti‐Mouse (715‐025‐151), used at 1 : 500.
8
Morphological Analysis of Aortic and Carotid Arteries
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