Cells were replated and grown (typically 48 h following drug exposure or introduction of small interfering RNA (siRNA; Sigma/Millipore), in RNAmax lipofection reagent (Thermo Fisher Scientific, Waltham, MA, USA), fixed in formaldehyde (4% v/v) and stained in a dark container with 0.1% w/v Thioflavin T. After four washes in PBS, cells were covered with Antifade + DAPI (Life Technologies, Grand Island, NY, USA) and fluorescence was captured using appropriate filters (DAPI/blue and Thioflavin T/green) with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope with motorized stage for automated well-by-well imaging. Immunohistochemistry methods were described previously (Balasubramaniam et al., 2018 (link)). Briefly, fixed cells were probed with mouse antibody to human Aβ [ab 11132 (AbCam), 1:400 dilution] for 2 h. After washes in PBS, cells were incubated 30 min at 22°C with goat anti-mouse secondary antibody coupled to Alexa488 (Life Technologies, 1:500 dilution) and imaged with a Keyence fluorescence microscope.
Antifade dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
Antifade DAPI is a laboratory reagent used to preserve fluorescent signals in microscopy applications. It contains a DNA-binding dye that labels nuclei and a chemical compound that helps prevent photobleaching of fluorescent dyes. Antifade DAPI is commonly used to stain and visualize DNA in fixed cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Ds fi2 camera, by Nikon (1 mentions)
Ab11132, by Abcam (1 mentions)
Alexa fluor 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Eight well chamber slide, by BD (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti lgr5, by Abcam (1 mentions)
Anti desmin, by Merck Group (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti α sma, by R&D Systems (1 mentions)
Anti vimentin, by R&D Systems (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
12 protocols using antifade dapi
1
Quantifying Amyloid-Beta Aggregation in Cells
2
Immunofluorescence Staining of Cell Cultures
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Cells cultured in eight-well chamber slides (Falcon, BD, Germany) and culture-inserts (ibidi) were washed twice with cold PBS, fixed with 4% para-formaldehyde for 15 min, permeabilised with 0.1 % Triton X-100 for 5 min, blocked with 5 % BSA, incubated with indicated primary antibodies: anti-GFAP and anti-desmin (Sigma, Darmstadt, Germany), anti-Lgr5 (Abcam, Cambridge, UK), anti-α-SMA, and anti-Vimentin (R&D, Minneapolis, MN, USA), APC-anti-CD271 (Miltenyi Biotech, Auburn, CA, USA) at 4 °C overnight and followed by anti-rabbit Alexa fluor 488 secondary antibody and anti-mouse Alexa Fluor 568-conjugated secondary antibody (Life technology, Darmstadt, Germany). The cells were then stained with anti-fade DAPI (Life Technology) for nuclear staining, and the images were acquired with an Olympus Axion microscope (Olympus, Tokyo, Japan).
3
Neuroblastoma Cells Protein Aggregation
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Exponentially growing cultures of SY5Y-APPSw (human neuroblastoma) cells were trypsinized and replated at 8000–10,000 cells/well in 96-well plates and grown for 16 h at 37 °C in DMEM + F12 (Life Technologies; Carlsbad, CA, USA) medium supplemented with 10% (v/v) fetal bovine serum. When cells reached ~40% confluence, siRNAs were transfected by lipofection (RNAiMax, Life Technologies; Carlsbad, CA, USA) to target genes encoding ANK3 (SAS1_Hs_00065571), YWHAZ (SASI_Hs01_00210839), YWHAG (SASI_Hs01_00201711), KIF5C (SAS1_Hs_ 00065571), PLEC (SAS1_Hs_00039321), or NUCL (SAS1_Hs_00217223), all obtained from Millipore-Sigma (St. Louis, MO, USA) and used as directed by the manufacturer. Transfected cells were assayed for protein aggregation 48 h later by fixation in 4% formaldehyde and staining with 0.1% w/v Thioflavin T. After four washes in PBS, cells were covered with Antifade + DAPI (Life Technologies) and fluorescence was captured in both blue and green channels (Keyence fluorescence microscope with motorized stage) for automated well-by-well imaging, with nine fields per well. Thioflavin T fluorescence intensity was divided by the number of DAPI+ nuclei to obtain normalized values (amyloid fluorescence per cell), summarized as means ± SD.
4
Micronuclei Formation Assay with EVs
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Cells (9 × 105) were cultured on autoclaved slides and incubated with 4 × 109 indicated EV preparations for 7 days (or as indicated). Slides were washed with PBS and fixed with 3.7% formaldehyde/PBS for 10 minutes at room temperature (RT) in a coplin jar. Slides were then washed again 3x with PBS for 5 minutes each at RT. Following this step, slides were serially treated with 70%, 90% and 100% dilutions of ethanol for 3 minutes each at RT and subsequently air dried. Antifade DAPI (Invitrogen, Catalog No. P36935) was added on the cover slips and placed on the slides before analyzing micronuclei formation.
5
Malaria Drug Screening Assay
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Iron (III) chloride hexahydrate, 98%, Iron (II) Chloride Tetrahydrate, ATA (2−amino−terephthalic acid), (NH4OH, 30% in water) were purchased from Alfa Aeser. o-Phenanthroline (Fischer scientific), 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), NHS-fluorescein-dextran (NHS-FITC), Antifade DAPI, RPMI 1640, Albumax I, LysoTracker Red DND-99 (Invitrogen: Carlsbad, CA, USA), Methanol, Ethanol, ethyl acetate (MERCK, India), L- Ascorbic acid (Vitamin C), Artesunate, hypoxanthine, Sodium bicarbonate, Sorbitol, Gentamycin, Giemsa stain, DMSO, low-melting-point agarose, normal melting point agarose, ethidium bromide, 2,4-Dinitrophenylhydrazine, Trichloroacetic acid, guanidine hydrochloride, Bovine serum albumin (Sigma Aldrich), percoll (GE, India), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thermo Fisher Scientific), RIPA buffer (Himedia Labs, India).
All chemicals were used as received without further purification.
All chemicals were used as received without further purification.
6
Quantitative Analysis of Actin Cytoskeleton
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Spheroids grown on 24-well ULA plates were collected and chemically dissociated using Accumax (Innovative Cell Technologies). Dissociated spheroid cells were plated on poly-L-lysine coated 25 mm coverslips (Fisher Scientific), and cells were left for 1 hour to adhere before fixation. Cells were fixed with 4% PFA for 10 min, washed with PBS for 10 min, permeabilized in 0.5% TritonX-100 in PBS for 5 min, blocked with 1% BSA and 0.05% Tween in PBS for 1 hour, stained with and ActinRed™ 555 ReadyProbes™ Reagent (Rhodamine phalloidin) (Thermofisher) for 1 hour, and mounted with anti-fade DAPI (Invitrogen). Images were acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss AG) with the 40X objective. Total fluorescence per cell was analyzed using ImageJ. The RGB image was converted to a grayscale 8-bit image type, the region of interest was selected (each cell), and then area, mean and integrated intensity were measured using the Analyze tool in ImageJ. Corrected fluorescent intensity (CTCF) for each cell was calculated according to the equation: CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings). Each field had at least 6 images of cells at 40X magnification.
7
Apoptosis and Necrosis Assay
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C4-2 and CWR22Rν1 cells were seeded on a two-chamber tissue culture glass slides and treated with 40μg/ml of MEM at 70% confluence for 24h. After washing with PBS cells were fixed with 2% paraformaldehyde followed by permeabilization with methanol and blocking with 2% serum. Incubation with primary antibodies overnight was followed by incubation with appropriate fluorophore tagged secondary antibodies. Antifade DAPI (Invitrogen, NY) was used as mounting and counterstaining medium. For analysis, Bio-Rad Radiance 2100 MP Rainbow system for biological imaging was used. To detect apoptotic and necrotic cells the Annexin-V-Fluos Staining Kit (Roche, Switzerland) was used according to the manufacturer’s protocol. Zeiss LSM 410 confocal microscopy was used to measured fluorescence. The cells stained with Annexin-V and unstained cells in a selected field were counted to ascertain the extent of apoptosis and necrosis.
8
Organoid Immunofluorescence Staining Protocol
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Organoids were dissociated from Matrigel using cell recovery solution (Corning) as aforementioned. Organoids were then processed as per published protocol57 (link). Organoids were stained with the following antibody: ADAR1 (1:100, Cell Signaling Technology, 14175), SCD1 (1:100, abcam, ab19862), and KHDRBS1 (1:1000, abcam, ab86239). Organoids were mounted and counterstained with antifade DAPI (Invitrogen).
9
Immunofluorescence Staining of Cells
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X (Sigma-Aldrich), blocked with 4% bovine serum albumen and incubated with primary antibody overnight at 4ºC before incubating with secondary antibody for 1 hr at room temperature. Cells were counterstained with anti‐fade DAPI (Invitrogen) and observed by fluorescent confocal microscopy (DM-RXA2, Leica). Images were processed with a MetaMorph Imaging System (Universal Imaging Corp).
10
Quantifying Lipid Droplets and ER Tracker
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Cells were incubated with 5 µg/mL BODIPY 493/503 (Invitrogen) or 1 µM ER tracker red (Invitrogen) for 1 h. Cells were fixed with 2% paraformaldehyde, mounted and counterstained with anti-fade DAPI (Invitrogen). Slides were imaged by confocal microscopy (Carl Zeiss) using Zen 3.0 software and quantified by ImageJ software. To quantify lipid droplets, the number of Maxima (point which emits saturated signal) was identified with an arbitrary threshold using ImageJ to determine the number of lipid droplets per image. The number of cells was determined by counting the number of DAPI-marked nucleus. The data is presented as the number of lipid droplet divided by the number of cells. To quantify ER tracker, the mean fluorescence intensity (MFI), which accounts for area and intensity of the target of interest, of the ER tracker is quantified using ImageJ.
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