Primary VS cells were seeded at 10,000 cells/well in SCM on 16-well culture slides precoated with 0.01% PLO and laminin at 5% CO2 and 37°C. After 24 h, cells were treated with CUDC907 (100 nM) or 0.0005% DMSO (vehicle) in D10 media. Cells were fixed, permeabilized and blocked for 2 h at RT. Slides were exposed to primary antibodies overnight at 4°C, secondary antibodies at RT for 2 h, and DAPI nuclear stain (4′,6-diamidino-2-phenylindole; ab104139, Abcam) for 15 minutes. Slides were cover-slipped with anti-fade mounting medium (Sigma). Primary antibodies used were rabbit anti-YAP (1:200; Cell Signaling, #14074) and rabbit anti-p21 (1:100; ThermoFisher, MA5-14949) and donkey anti-rabbit IgG conjugated to AlexaFluor 594 (1:200; ThermoFisher). Images were obtained with the Leica SP5 Inverted Confocal Microscope (40X oil immersion lens) and assessed qualitatively.
Anti fade mounting medium
Manufactured by Merck Group
Sourced in Sao Tome and Principe, United States
Anti-fade mounting medium is a lab equipment product designed to preserve and protect fluorescent signals in microscopy samples. It is formulated to reduce photobleaching and maintain the integrity of fluorescent labels during long-term storage and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Edta containing antigen retrieval solution, by Merck Group (2 mentions)
Dm2000 fluorescent microscope, by Leica (2 mentions)
Ab104139, by Abcam (1 mentions)
Donkey anti rabbit igg conjugated to alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Sp5 inverted confocal microscope, by Leica (1 mentions)
Rabbit anti yap, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
A1rsi confocal microscope, by Nikon (1 mentions)
Acridine orange solution, by Merck Group (1 mentions)
Alexa 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Deadend colorimetric tunel system, by Promega (1 mentions)
Developer xd 1, by Definiens (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Phalloidin tritc conjugate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
9 protocols using anti fade mounting medium
1
Analyzing YAP and p21 in Primary VS Cells
2
Immunofluorescence Staining of Cells
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Cells were seeded onto glass coverslips and treated as described. At indicated timepoints, samples were fixed with 4% paraformaldehyde at room temperature for 10 min before permeabilization with 0.1% Triton-X at room temperature for 5 min. The fixed samples were rinsed blocked with 1% bovine serum albumin solution at 37 °C for 1 h and incubated with primary antibodies overnight at 4 °C before three consecutive washes in PBS to remove excess antibodies. Incubation with secondary conjugated antibodies was carried out for 1 h at 37 °C before coverslips are washed and mounted onto a glass slide with anti-fade mounting medium containing DAPI (Sigma-Aldrich). Slides were imaged using Nikon A1R+si confocal microscope at Nikon Imaging Center (Singapore Bioimaging Consortium).
3
Visualizing Extracellular Vimentin in Tissues
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To evaluate presence of extracellular vimentin on the surface of cells within human tissue, fluorescent immunohistochemistry staining was performed. To do so, paraffin-embedded human lung and fat tissue, as well as sputum obtained from cystic fibrosis patients were cut into 15 μm thick slices on a rotary microtome and placed on glass slides. After overnight drying, slides were rehydrated by immersion in xylene and ethanol for 10 min at each step. Slides were hydrated with PBS and transferred to EDTA-containing antigen retrieval solution (Sigma) for 1h at 100°C. Subsequently, samples were blocked in blocking buffer (1% BSA in PBS) for 30 min at RT. Primary rabbit anti-vimentin antibodies to the C-terminus at 1:200 were placed directly on tissues and left for overnight incubation in humidified chamber at 4°C. Afterwards, slides were washed three times for 15 min in PBS and incubated with secondary anti-rabbit Alexa Fluor 488-conjugated antibody at 1:500 for 1 h at RT, protected from light. Following the washing step, tissues were counterstained with DAPI and mounted with anti-fade mounting medium (Sigma). Visualization was performed on Leica DM2000 fluorescent microscope
4
Lysosomal Integrity Assay in Mouse Hepatocytes
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In order to perform lysosomal integrity assay, primary cultured mouse hepatocytes were first preincubated with 5 μg/mL acridine orange (AO) solution (Sigma Aldrich, St. Louis, MO, U.S.A) in William's E medium for 20 min in dark at 37°C [19 (link)]. Following staining, the cells were rinsed twice with PBS to remove the excess stain and treated with or without 10 μM arsenic for different time points. At the end of treatment, the cells were mounted on a glass slide with a cover slip using antifade mounting medium (Sigma Aldrich, St. Louis, MO, U.S.A) and observed under the confocal microscope using excitation wavelength of 488 nm and emission wavelength of 535 nm.
5
Immunostaining of Collagen I in Cells
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Cells were fixed with acetone: methanol (1:1) and air‐dried for 20 min. The cells were rehydrated with cold PBS and incubated with collagen I primary antibody (1:100) for 1 h at room temperature. Cells were then incubated with Alexa‐488‐labeled secondary antibody (1:100) for 1 h at room temperature. Thereafter, the cells were fixed with DAPI‐containing antifade mounting medium (Merck, Sigma). The staining was visualized, and images were captured using fluorescence microscopy (Evos).
6
Immunohistochemical Staining of Retinal Photoreceptors
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The retinal sections were permeabilized using 5% donkey serum in PBS containing 0.2% Triton X-100, as described previously. The sections were incubated with primary polyclonal anti-rhodopsin antibody (1 : 900, VPP, custom made [12 (link)]) and polyclonal anti-S-opsin antibody (1 : 500, Millipore, USA) at 4°C overnight. Alexa488-conjugated goat anti-rabbit secondary antibody (ThermoFisher Scientific, USA) was diluted at 1 : 300 and incubated at room temperature for 1 h. The sections were mounted with an anti-fade mounting medium (Sigma, USA). All the antibodies were diluted in 5% donkey serum prepared in 1XPBS supplemented with 0.2% Triton X-100. The images were acquired by a Zeiss LSCM 900 confocal microscope.
7
Quantifying Apoptosis in Lung Tissue and MBECs
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TUNEL analysis was conducted using an In Situ Cell Death Detection kit (DeadEnd™ Colorimetric Tunel System; Promega Corporation). For lung tissue, tissue sections (4 µm) were deparaffinized using xylene, rehydrated in graded ethanol, and rehydrated for 3 min. The sections were then incubated with TUNEL (DeadEnd™ Colorimetric Tunel System; Promega Corporation ) for 2 h at 37˚C according to the manufacturer's protocol. For MBECs, cells were treated with 4% paraformaldehyde for 15 min at room temperature. Cells were then washed with PBST three times at room temperature and incubated with TUNEL (DeadEnd™ Colorimetric Tunel System, Promega) for 1 h at 37˚C according to the manufacturer's protocol. Cells were washed with PBS three times at room temperature and then incubated with 5% DAPI (Sigma-Aldrich; Merck KGaA) under Antifade mounting medium (cat. no. P0126; Beyotime Institute of Biotechnology) for 30 min at 37˚C. Finally, images of sections and cells were captured in six fields of view with a ZEISS LSM 510 confocal microscope at 488 nm at a magnification of x100. The apoptosis rate was measured using Developer XD 1.2 software (Definiens AG).
8
Extracellular Vimentin Detection in Human Tissues
9
Visualizing Viral Antigen and Actin Cytoskeleton
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After washing in PBS, cells were fixed in 3.7% paraformaldehyde/PBS (Sigma-Aldrich) for 10 min at room temperature and suspended in cold acetone (-20 o C) for 5 min. Before staining, fixed cells were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich) for 30 min at room temperature. Filament structures of actin were visualised by TRITC-phalloidin conjugate (500 ng/ml; Sigma-Aldrich). The presence of viral antigen was detected by means of direct immunofluorescence, using polyclonal antiserum EHV-1/ERV conjugated to FITC (VMRD, Inc.). Cell nuclei were stained with Bisbenzimidine/Hoechst 33258, in compliance with manufacturer's recommendations. Afterwards, coverslips were mounted on microscope slides using anti-fade mounting medium (Sigma-Aldrich). Confocal microscope images were obtained using Leica SP8-WLL white-light laser confocal microscope under 63x oil-immersion lens and analysed with LAS AF Lite 3.2.0 and Adobe Photoshop software.
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