hSAECs and SMARCA4 KD cells were plated on coverslips or transwell membrane. Human primary fibroblasts were plated on coverslips in the lower compartment of transwell chamber. After virus infection, with or without 5 µM MMP-9 inhibitor I (Santa Cruz, CAS 1177749-58-4), cells were fixed with 4% paraformaldehyde, permeablized with 0.1% Triton X-100, blocked with 5% goat serum and incubated with primary antibody overnight. Primary antibodies used were anti-Vimentin (Sigma, V6630); anti-E-Cadherin (cell Signaling, 3195); anti-Ki67 (Thermo, MA5-14520). On second day, Alexa-fluor goat secondary antibody and/or phalloidin (Thermo, A12380) were added. After 1 h, cells were washed and mounted using Prolong Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Molecular Probes). To assay syncytia formation, fixed cells were stained with CellBrite (Biotium, 30090, 5 ul/1ml PBS), and mounted with Antifade Mountant containing DAPI. The cells were visualized with ECHO Revolve fluorescent microscope.
Prolong diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) is a mounting medium designed for fluorescence microscopy. It contains an antifade agent to reduce photobleaching and DAPI, a fluorescent dye that binds to DNA, providing nuclear staining.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Mmp 9 inhibitor 1, by Santa Cruz Biotechnology (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Te2000 e inverted microscope, by Nikon (1 mentions)
Ds ri, by Nikon (1 mentions)
Nigericin, by Merck Group (1 mentions)
Bee venom, by Merck Group (1 mentions)
Anti asc n 15 r, by Santa Cruz Biotechnology (1 mentions)
Lps escherichia coli o55 b5, by Merck Group (1 mentions)
Dp controller software, by Olympus (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Ab10812, by Abcam (1 mentions)
Ab85454, by Abcam (1 mentions)
20 protocols using prolong diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi
1
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition
2
Immunocytochemistry of Cardiomyocyte Rac1 and zDHHC3
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Immunocytochemistry was performed on adult cardiomyocytes in suspension exactly as described previously 61, 64 . Cardiomyocytes were isolated from mouse hearts by Langendorff perfusion, fixed with 4% paraformaldehyde for 15 minutes at room temperature, incubated in blocking solution (PBS, 5% goat serum, 1% BSA, 1% glycine, 0.2% triton X-100) for one hour at room temperature and then immunostained with Rac1 (BD Transduction Laboratories, #610650) or zDHHC3 (Abcam, #ab31837) antibodies. Primary antibodies diluted 1:50 in blocking solution overnight at 4°C. Cardiomyocytes were then washed in PBS with 0.1% NP-40, incubated with Alexa Fluor secondary antibodies (Molecular Probes) diluted 1:1,000 in blocking solution for two hours at room temperature, washed again in PBS with 0.1% NP-40, and mounted on slides with Prolong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). Imaging was performed using a Nikon A1 Confocal microscope.
3
Immunocytochemistry and Morphometric Analysis
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Cells fixed in 4% paraformaldehyde at 4°C for 10 min were washed in 1× phosphate-buffered saline (PBS), and then permeabilized in PBS with 0.5% Triton X-100 for 5 min at room temperature (RT). Cells were then blocked with 3% goat serum in PBS containing the primary antibody TUJ1 (anti-rabbit, 1:500; Abcam) and incubated overnight at 4°C. Cells were then washed with PBS and incubated with Alexa FluorTM goat anti-rabbit 594 sary antibody for 1 h at RT. Individual cell nuclei were confirmed using Prolong Diamond antifade mountant with 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies). Imaging was undertaken on a Nikon TE2000E inverted microscope (Nikon) equipped with a Nikon DS-Ri camera. Images were converted to greyscale in ImageJ, scaled and the cell soma size measured using the polygonal selection tool. Neurite number, neurite length and the number of branch points were determined using the NeuronJ plugin in Fiji. Morphological characterization was undertaken from 30 cells per donor age group.
4
Inflammasome Activation Mechanisms
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The following reagents were used in this study: Escherichia coli LPS O55:B5, nigericin and bee venom (Sigma-Aldrich, Madrid, Spain); caspase-8 inhibitor II X-IETD-FMK (Merck-Millipore, Darmstadt, Germany); fluorogenic substrates for caspase-1 YVAD-AFC, caspase-3 DEVD-AFC and caspase-8 IETD-AFC (PromoKine, Heidelberg, Germany); crosslinking reagent SDA (Thermo Scientific, Waltham, MA, USA); rabbit polyclonal antibody against IL-1β, caspase-1 p10 (M-20) and anti-ASC (N-15)-R (Santa Cruz Biotechnology, Dallas, TX, USA); ECL horseradish peroxidase-conjugated secondary antibody for immunoblot analysis (GE Healthcare, Uppsala, Sweden); and Alexa Fluor 647-conjugated donkey anti rabbit IgG secondary antibody and ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Paisley, UK).
5
Amniotic Fluid Bacterial Viability Assay
6
Immunofluorescence Analysis of KDM4A, KDM4C, and H3K9 Trimethylation in TNBC Cells
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Triple-negative breast cancer cells were grown on coverslips and treated with NCDM-32b as previously described. After 48 hours, the samples were pre-extracted with 0.2% PBS/Triton X-100 and fixed in 3.7% formaldehyde for 15 minutes. The fixed cells were rendered permeable with 0.5% Triton X-100 in PBS and then incubated with 3% bovine serum albumin in PBS for one hour. Next, the cells were treated with anti-KDM4A (Abcam ab24545), anti-KDM4C (Abcam ab85454), or anti-trimethyl H3K9 (Abcam ab10812) antibody overnight at 4°C at 1:250 dilution, followed by three washes with PBS containing 0.01% Triton X-100 and the secondary antibody incubation (Alexa Fluor 488, 1:500 dilution, Abcam 150077) for one hour. Finally, the cells were washed twice and mounted in ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies). All images were obtained on an Eclipse Ni-E microscope (Nikon) and analyzed with ImageJ software (National Institutes of Health).51 (link)
7
Immunofluorescence imaging of zebrafish liver and LX-2 cells
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Adult zebrafish livers were dissected and fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) overnight at 4°C. Livers were incubated in 10%, 20%, and 30% sucrose in PBS for 24 hours each at 4°C. Samples were processed using a published protocol.[8 ] LX‐2 cells were seeded at 200,000 cells/mL on chamber slides (Nunc Lab‐Tek; ThermoFisher), grown overnight, fixed in 4% paraformaldehyde for 15 minutes, permeabilized with PBS + 0.4% Triton X‐100, and blocked with PBS + 0.25% Triton X‐100 + 5% FBS + 2% bovine serum albumin for 1.5 hours at room temperature. Samples were stained with primary antibody (Table S1 ) overnight at 4°C and followed by secondary antibody for 1.5 hours in the dark at room temperature. Samples were mounted with ProLong Diamond Antifade Mountant with 4´,6‐diamidino‐2‐phenylindole (DAPI; Life Technologies) and imaged with a Leica SP5 DMi.
8
Tau Localization in HEK293T Cells
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HEK293T wild-type and HEK293T NDUFA11 knockout cells were transfected with BiFC plasmids that encoded Tau for 72 h, washed twice with PBS, and fixed with 3.7% formaldehyde for 10 min at 4°C. The cells were then washed twice with PBS and permeabilized for 5 min by treatment with 0.1% Triton X-100 in PBS. They were then rinsed once with PBS and once with water, and the samples were mounted in ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific; catalogue no. P36962). The analysis was performed using a Zeiss LSM700 confocal microscope.
9
Visualizing Newly Synthesized RNAs
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Newly synthesized RNAs were labeled by including 2 mM 5-ethynyl uridine (5-EU) into the cell culture medium, and modified incorporated nucleotides were reacted with an azide-conjugated fluorophore, Alexa Fluor 488, by following the manufacturer’s protocol for the Click-iT RNA Alexa Fluor 488 imaging kit (Thermo Fisher Scientific). Cell nuclei were stained with ProLong diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Scientific). Samples were imaged using a confocal setup (Zeiss Airyscan equipped with a 63× objective with an NA of 1.3), and the images were processed using ZEN lite software.
10
Immunostaining Protocol for Lysosomal Marker LAMP1
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Immunostaining for LAMP1 was used as a lysosomal marker in the proximal tubules. After deparaffinization of the paraffin sections, they were heated at 100°C for 20 min in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. The sections were incubated with 4% Block ACE (DS Pharma Biomedical, Osaka, Japan) in PBS (–) containing 0.05% Tween 20 (PBS-T) for 2 h at 25°C, then washed three times with PBS-T. Primary antibodies against LAMP-1 (ab25245, dilution 1:50; Abcam, Cambridge, UK) and aquaporin 1 (AB2219, dilution 1:300; Merck Millipore) were used as proximal tubule markers. The highly cross-adsorbed secondary antibodies Alexa fluor 488 conjugated donkey anti-rat IgG (H+L) (AB_2535794, 1:500 dilution; Thermo Fisher Scientific) or Alexa fluor 555 conjugated donkey anti-rabbit IgG (H+L) (AB_2535792, 1:500 dilution; Thermo Fisher Scientific) were then used. ProLong™ Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) was used for mounting. Fluorescent images were obtained using confocal microscopy (LSM710; Carl Zeiss) equipped with a microscope (Axiovert 200M; Carl Zeiss) with a Plan Apochromat 63×/1.4 NA oil immersion lens, and the images were visualized using the ZEN software (Carl Zeiss).
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