HUVECs were seeded on coverslips and incubated in the presence of MAPF for 8 h. Cells were then rinsed with PBS and fixed with 10% v/v formalin in PBS (pH 7.4). A blocking solution (5% milk in 0.1% v/v Triton X-100) was applied to prevent nonspecific binding. Primary antibody against VE-cadherin (Cell Signaling Technology, USA) and β-catenin (BD Bioscience, USA) in blocking buffer was incubated with the HUVECs at 4 °C overnight. After the antibody was washed, the slides were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich) for 1 h. Finally, coverslips were mounted with mounting medium (Gel Mount Aqueous, Sigma) and photographed with a Nikon D1X digital camera (Carl Zeiss, Oberkochen, Germany).
Gel mount aqueous
Manufactured by Merck Group
Sourced in Germany
Gel Mount Aqueous is a laboratory mounting medium designed for use with gel electrophoresis techniques. It is a water-based solution that aids in the preservation and display of gel samples. The product functions to mount and protect gel samples, enabling clear visualization and documentation of electrophoresis results.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate conjugated goat anti mouse and anti rabbit igg, by Merck Group (2 mentions)
Ve cadherin, by Cell Signaling Technology (2 mentions)
Fitc conjugated goat anti mouse igg, by Merck Group (2 mentions)
β catenin, by BD (1 mentions)
F actin, by Merck Group (1 mentions)
Phalloidin, by Merck Group (1 mentions)
6 protocols using gel mount aqueous
1
Immunofluorescence Analysis of HUVEC Junctions
2
Immunofluorescence Imaging of HUVEC Cells
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HUVECs were seeded on coverslips and incubated in the presence of LCPF for 8 h. Cells were then rinsed with PBS and fixed with 10% v/v formalin in PBS (pH 7.4). A blocking solution (5% milk in 0.1% v/v Triton X-100) was applied to prevent nonspecific binding. Primary antibody against VE-cadherin (Cell Signaling Technology, USA) in blocking buffer was incubated with the HUVECs at 4 °C overnight. After the antibody was washed, the slides were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich) for 1 h. Finally, coverslips were mounted with a mounting medium (Gel Mount Aqueous, Sigma) and photographed with a Nikon D1X digital camera (Carl Zeiss, Oberkochen, Germany). For filipin cholesterol staining, HUVECs were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min and stained with 50 μg/mL filipin in PBS at room temperature for 2 h. Cells were then washed with PBS three times and mounted.
3
Immunofluorescence Staining of Endothelial Cells
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After incubation with MAPF, HUVEC on coverslips were rinsed with PBS and then fixed with 10% formalin in PBS (pH 7.4). A blocking solution (5% milk in 0.1% Triton X‐100) was used to block nonspecific binding sites. Then, a primary antibody against p‐paxillin (BD Bioscience) in blocking buffer was incubated with the HUVEC at 4°C overnight. The next day, F‐actin and FITC‐conjugated goat anti–mouse IgG (both from Sigma‐Aldrich) were applied for 1 hour. DAPI was used for counterstaining. Finally, the cells were mounted with mounting medium (Gel Mount Aqueous, Sigma) and recorded with a Nikon D1X digital camera (Carl Zeiss, Pberkochei).
4
Filipin Staining of Bladder Cancer Cells
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After different treatments, human bladder cancer cells were seeded on coverslips and incubated overnight. The cells were rinsed with PBS and fixed. After washing with a wash buffer, Filipin III solution (Abcam, Boston, MA, USA) was added and incubated for 1 h. Then, the cells were mounted with mounting medium (Gel Mount Aqueous, Sigma), and images were captured by a digital camera Nikon D1X (Carl Zeiss, Oberkochen, Germany).
5
Immunofluorescence Staining of Monocytes, Macrophages, and RANK
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To verify the FACS results following 1 day of OTM and RvD1 administration, IF staining was performed. Briefly, the cryo-sectioned slides (detailed above) were washed in PBS, embedded in warm antigen‐retrieval citrate buffer (Abcam, Cambridge, MA) for 20 min and washed three times with cold PBS. Next the slides were washed with Tris buffer (TBS) containing 0.025% Triton X‐100 and blocked in 1% bovine serum albumin (BSA) in TBS for 1 hour at room temperature. Then the slides were incubated overnight at 4°C with primary antibodies: Monocyte (CD64), Macrophage (F4/80) and RANK (CD265) in PBS containing 1% BSA (all purchased from Abcam). Samples were incubated with a secondary antibody conjugated to a fluorophore –Donkey Anti-Rat IgG (purchased from Abcam) in PBS with 1% BSA for 1 hour at room temperature, washed three times with TBS and counter‐stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI). The samples were sealed with Gel Mount Aqueous (Sigma‐Aldrich). Negative staining controls included slides from which the primary antibody was omitted. The samples were analyzed under a fluorescence microscope (Nikon TL, Tochigi, Japan).
6
Immunofluorescence Analysis of HUVEC Cytoskeleton and Proliferation
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Human umbilical vein endothelial cells were seeded on coverslips and incubated in the presence of BAPF for 24 hours. Cells were then rinsed with PBS and fixed with 10% formalin in PBS (pH 7.4). A blocking solution (5% milk in 0.1% Triton X‐100) was applied to prevent nonspecific binding. Primary Ab against p‐paxillin (BD Biosciences) in blocking buffer was incubated with the HUVECs at 4°C overnight. After the Ab was washed, the slides were incubated with phalloidin (for F‐actin staining) and FITC‐conjugated goat anti‐mouse IgG (both from Sigma‐Aldrich) for 1 hour. 4'‐6‐Diamidino‐2‐phenylindole was used for nuclear staining. Finally, the cells were mounted with mounting medium (Gel Mount Aqueous; Sigma) and photographed with a Nikon D1X digital camera (Carl Zeiss). The same protocol was carried out with a primary Ab targeting Ki‐67 (Genetex) in place of the p‐paxillin Ab to quantify proliferative activity.
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