Human embryonic kidney (HEK-293) cells (ATCC) were plated on 50 µg/ml poly-L-lysine (Sigma Aldrich Inc.)-coated coverslips (Fisherbrand, Inc.) in 24-well plates (JetBiofil) and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) supplemented with 5 mg/ml penicillin-streptomycin (Hyclone). Cells at 80% confluency were transfected with 0.5 µg of GFP-CASK-WT and GFP-CASKL209P DNA per well using the calcium phosphate method. Twenty hours post-transfection, cells were washed twice with phosphate buffered saline (Sigma Inc.) and fixed for 15 minutes at room temperature using a 4% paraformaldehyde solution. Coverslips were mounted on microscope slides (Premiere) using Vectashield (Vector Laboratories Inc.) and visualized using confocal laser scanning microscopy (ZEISS Axio Examiner.Z1 LSM 710). The percentage of transfected cells with visible aggregates was counted using the cell counter plugin of the Image J program. For each condition, five high-power field images were analyzed for aggregation. Total cells and cells containing aggregates were visually identified and tallied. Percent of total cells containing aggregates was then calculated. The image analysis procedure was repeated 3 times for each condition and averaged.
Microscope slides
Manufactured by Vector Laboratories
Sourced in Germany
Microscope slides are transparent, thin glass or plastic sheets used to hold samples for microscopic examination. They provide a stable, flat surface to place and observe specimens under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
24 well plate, by Jet Biofil (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Coated coverslips, by Thermo Fisher Scientific (1 mentions)
Hek293 cells, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Model axioimager m1, by Zeiss (1 mentions)
Canada balsam, by Junsei (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
12 well plates, by Iwaki (1 mentions)
5 protocols using microscope slides
1
Imaging of CASK Aggregation in HEK-293 Cells
2
Immunofluorescence Analysis of YAP in Cell Lines
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TE-8 and TE-12 cells were seeded onto glass coverslips and further treated with LY294002 with or without UA (20 μM) plus DIM (50 μM) for 48 h in a six-well plate. Cells were rinsed with DPBS twice and fixed with 4% methanol in DPBS for 40 min. Then, 0.1% Triton X-100 (Sigma-Aldrich) in DBPS was added for 10 min for permeabilization. After blocking, anti-YAP antibody (dilution 1:100) was added to cover glasses and stored at 4°C overnight. Cover glasses were rinsed, and rabbit secondary antibody (dilution 1:100) was added at room temperature for 1 h in the dark. Cells were stained with DAPI (dilution 1:500) for 5 min at room temperature in the dark. Cover glasses were attached to microscope slides (Paul Marienfeld GmbH & Co. KG) with mounting solution (Vector Laboratories, Inc.), and the edges were fixed with Canada balsam (Junsei Chemical Co.). Images were obtained under a fluorescence microscope (Zeiss Model Axio Imager M1). Fluorescence values were detected and merged using ImageJ.
3
Immunofluorescence Staining of Epithelial Cells
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Epithelial cells after the first passage were seeded in M199 with P/S and 10% NCS onto 8-chamber cell imaging coverglasses (Eppendorf, Germany) and cultured until reaching 70% confluence. Cells were washed three times with pre-warmed PBS and fixed in 4% paraformaldehyde (pH 7.45) for 20 min. Non-specific binding was blocked by 1 h of incubation in blocking buffer (0.1 M PBS, 0.1% BSA, 0.05% trimerosol) with 10% Normal Donkey Serum (Sigma Aldrich, Germany). Overnight incubation with primary antibodies was performed at 4 °C (Supplementary data 2 ). After washing in PBS, secondary antibodies were added into each well (donkey anti-rabbit antibodies conjugated to Alexa 488, Life Technologies, USA), and a 90-min incubation was performed in the dark. Then, washing in PBS was repeated three times, and cells were incubated in 0.01% DAPI (Sigma Aldrich, Germany) for 30 min. The plastic chamber was removed from each coverslip, which were mounted onto microscope slides (Menzel, Germany) using VECTASHIELD® Antifade Mounting Medium (Vector Laboratories, UK). Negative controls without primary, secondary and both antibodies as well as isotype controls were also included. Pictures were taken with an LSM 800 confocal microscope (63×/1.40 oil objective) (Zeiss, Germany).
4
Immunostaining of Telomerase Reverse Transcriptase
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Cells were seeded on cover slips (19 mm Ø, VWR, Lutterworth, Leicestershire, UK) in 12-well plates (IWAKI, Tokyo, Japan), treated as described above and fixed with 4% PFA. Cells were blocked and permeabilised with PBG-Triton (0.2% cold water fish gelatine, 0.5% BSA, 0.5% Triton X-100 in PBS), followed by 2 h of incubation with the primary antibody to telomerase reverse transcriptase, 1:2000, (Abcam/Epitomics UK, 32020). Afterwards cells were washed twice with PBG and incubated with the secondary antibody, IgG -goat-anti rabbit Alexa-Fluor 594, 1:2000 (Invitrogen, Inchinnan, UK) for 1 h, followed by PBS washes. Nuclear counterstain was performed with DAPI (CyStain UV Ploidy, Partec, Muenster, Germany) and the coverslips then mounted on Microscope Slides using Vectashield anti-fade mounting medium (Vector Laboratories, Peterborough, UK). Slides were examined with a Zeiss Axio Imager Z1 (Carl Zeiss Ltd., UK and AxioVision 4.8.2 (Carl Zeiss Ltd., Cambourne, UK) imaging software.
5
Chemotactic Assay for rBM-MSCs
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The rBM-MSCs were seeded onto 8.0-mm pore size poly(ethylene terephthalate) track-etched membrane cell culture filter inserts (Becton Dickinson, Franklin Lakes, NJ). The filters were incubated in a 24-well plate containing 10% fetal bovine serum DMEM. After the adhesion of the cells on the filters, the filters were transferred to a new 24-well cell culture plate containing one of five chemotactic agents. Serum-free DMEM was then added on top to each filter, and the filters were incubated in hypoxia for 16 hours. Afterwards, the cells were fixed with 3.7% paraformaldehyde, permeabilized with 100% methanol, and stained with 4,'6-diamidino-2phenylindole. A cotton swab was then used to disrupt the cells on the filters. The filters were carefully cut and mounted on microscope slides with the bottom facing up (Vector Labs, Burlingame, CA). Fluorescence microscopy was performed at original magnification Â20.
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