bEnd.3 cells were cultured on gelatin-coated coverslips, and ihBMECs were grown on collagen/fibronectin-coated coverslips. Before adding acetylated LDL, cells were incubated with medium supplemented with 0.3% bovine serum albumin (BSA, Fisher Scientific, Waltham, MA, USA) for 1 h at 37°C to block non-specific binding. Subsequently, bEnd.3 cells and ihBMECs were incubated with 10 μg/mL Alexa Fluor 488-conjugated acetylated LDL (Invitrogen, Carlsbad, CA, USA) for 4 h at 37°C followed by three PBS washes. After fixation with 4% paraformaldehyde, the nucleus was stained with DRAQ5 (Biolegend, San Diego, CA, USA), and coverslips were mounted with UltraCruz Aqueous Mounting Medium (Santa Cruz Biotechnology, Dallas, TX, USA). Three random fields per coverslip (of approximately 50 cells each) were imaged using a 63x oil immersion objective with a Leica SP5 confocal microscope (Leica, Wetzlar, Hessen, Germany) and manually quantified for cells positive with acetylated LDL uptake. iPSC IMR90-4 cells growing on Matrigel-coated coverslips were stained for acetylated LDL uptake as a phenotype negative control.
Ultracruz aqueous mounting medium
Manufactured by Santa Cruz Biotechnology
Sourced in United States
UltraCruz Aqueous Mounting Medium is a water-based solution for mounting microscope slides. It is designed to preserve the clarity and integrity of biological samples during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Draq5, by BioLegend (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Ab150079, by Abcam (1 mentions)
Tcs sp8, by Leica (1 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Occludin, by Cell Signaling Technology (1 mentions)
Claudin 2, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Ab109497, by Abcam (1 mentions)
Fv1000, by Zeiss (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
5 protocols using ultracruz aqueous mounting medium
1
Acetylated LDL Uptake Assay in Cells
2
Immunofluorescence Staining of COL12A1 in iCCA Cells
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A total of 2 × 103 iCCA cells, seeded on coverslips coated in a 24-well cell culture plate, were incubated in RPMI-1640 medium containing 10% FBS and allowed to adhere for 6–8 h. After 3 times washing using phosphate-buffered saline (PBS), cells were fixed with 4% paraformaldehyde for 10 min at room temperature, then washed 3 times with ice-cold PBS, permeabilized with 0.1% Triton-X100 in PBS for 10 min, blocked with 1% bovine serum albumin (BSA) and 22.52 mg/mL glycine in PBS supplemented with 0.1% Tween 20 (PBST) at room temperature for 30 min, and finally incubated with anti-COL12A1 antibody (1:50) overnight at 4 °C in the dark. After 3 times washing using PBS, cells were then incubated with fluorescence-labeled secondary antibody (1:200, Abcam, #ab150079) for 1 h in the dark, washed 3 times with PBS, followed by incubation with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 1 min in the dark, and finally rinsed 3 times with PBS in the dark. Coverslips were mounted with UltraCruz® Aqueous Mounting Medium (sc-24941, Santa Cruz) and sealed with nail oil. Finally, cells on the coverslip were microphotographed using a Leica fluorescence microscope (TCS SP8, Germany).
3
Transfection of AD293 Cells with siGLO
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AD293 cells were seeded at 5 × 104 cells per well on a 24-well plate with coverslips and grown in a cell incubator at 37°C with 5% CO2 overnight. Transfection experiments were performed 24 h later. The siGLO samples were diluted in OptiMEM reduced-serum cell culture medium (ThermoFisher) and added to the cells in duplicates at final 100 nM siGLO concentration (50 pmol–665 ng per 500 μL growth medium per well). As a positive control, 50 pmol siGLO was complexed with 1 μL LPF and used for transfection at same 100 nM final siGLO concentration. After 24 h of incubation at standard culture conditions, the cells were washed with PBS (Sigma Aldrich), fixed with 4% paraformaldehyde (ThermoFisher) in PBS, and counterstained with DAPI in UltraCruz Aqueous Mounting Medium (Santa Cruz Biotechnology). Imaging was performed using Zeiss Model AxioScope A.1 LED Fluorescent Illumination Microscope equipped with DAPI (365 nm) and GFP (470/40 nm) filter and Zeiss AxioCam MRc digital camera (Carl Zeiss).
4
Immunofluorescent Staining of Epithelial Junctions
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After bacteria-cell co-culture, Caco-2 cell monolayers were washed with PBS before being fixed with 100% ice-cold methanal at -20 °C for 5 min and permeabilized with 0.5% Triton X-100 in PBS room temperature for 10 min. The monolayers were then washed with PBS and blocked for 1 hour with 4% (w/v) bovine serum albumin (BSA) at room temperature. The cells were then incubated with either antibody of Zo-1(Cell Signaling Technology,13663S), Occludin (Cell Signaling Technology, 91131S), or E-cadherin (Cell Signaling Technology, 3195S). Cells were fixed with 4% paraformaldehyde when staining the Claudin-2 with Claudin-2 (Invitrogen, 51–6100) primary antibodies. The cells were rinsed again with washing PBS three times, followed by incubation with the Alexa Fluor 594 goat anti-rabbit secondary antibody (Cell Signaling Technology, 8889S) for 1 h at room temperature. The cells were rinsed again with PBS before the membranes were separated from the Transwell insert using a scalpel. The membrane was finally mounted with cell side up between a slide and coverslip with UltraCruz® Aqueous Mounting Medium (Santa Cruz Biotechnology). The mounting media contains DAPI to stain all the nuclei. The microscopy of the mounted membranes was performed on a Nikon Confocal Laser Scanning Microscope.
5
Immunofluorescence Staining of TSPO in Tissues
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Immunofluorescence staining was performed as previously described [12 (link)]. Briefly, tissue cryosections or collected blastocysts at E3.5 were fixed in 4% paraformaldehyde, permeabilized with 0.1% triton X, blocked with 1% bovine serum albumin containing 1% donkey serum, and incubated in primary rabbit anti-TSPO monoclonal antibody (EPR5384) (ab109497; Abcam) [18 ] followed by secondary donkey antirabbit immunoglobulin G (H + L) antibody conjugated with Alexa Fluor 546 (Thermo Fisher) [19 ]. Nuclei were counterstained using UltraCruz aqueous mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI; Santa Cruz Biotechnology Inc). Microscopy was performed using Olympus FV1000 and Zeiss LSM 780 confocal laser scanning microscopes and an inverted Olympus microscope for epifluorescence imaging. All images were analyzed using ImageJ software (National Institutes of Health).
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