SLOS and control cells were cultured on cover slips and fixed using 10% formalin buffer. After 30 minutes of blocking in 3% BSA in PBS, the cover slips were incubated overnight at 4°C with primary antibodies for LC3B & actin at 1:200 or primary antibodies for Parkin or Pink-1 (Abcam, Cambridge, MA) at 1:300 overnight at 4°C. Cells were washed three times in PBS and incubated with corresponding secondary antibodies (Cy3 or FITC conjugated anti-rabbit or anti-mouse antibodies) at 1:400 for 1 hour. Cells were washed again with PBS and mounted in a medium containing 4′-6-diamidino-2-phenylindole (DAPI) to stain the nuclei (Vector Laboratory, Burlington, Ontario, Canada). Slides were viewed using a Confocal Laser Scanning Biological Microscope (Olympus, Center Valley, PA). Images were captured using Fluoview FV1000 software. A negative control to confirm the specificity of the immunostaining was prepared using non-immune goat serum in place of the primary antibody.
Confocal laser scanning biological microscope
Manufactured by Olympus
Sourced in Japan
The Olympus Confocal Laser Scanning Biological Microscope is a high-resolution imaging system designed for detailed analysis of biological samples. It utilizes a focused laser beam and a pinhole aperture to capture optical sections, enabling the reconstruction of 3D images from the collected data.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Fluoview ver 1.7a viewer, by Olympus (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 protocols using confocal laser scanning biological microscope
1
Immunofluorescence Analysis of Autophagy Markers
2
Daphnetin Modulates NFAT and NF-κB Localization
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The nuclear distribution of NFAT and NF-κB were measured using immunofluorescense. Mouse CD3 T cells (2×106 cells/mL) in 2 mL of RPMI 1640 complete medium were incubated with 1 mL daphnetin (4, 8, 16 µg/mL) in 6-well plates at 37°C for 1 h followed by 0.5 h incubation with 1 mL of ConA (5 µg/mL), resulting in a final well volume of 4 mL per well. After over-night incubation in anti-NFAT2 antibody (1∶350) or anti-NF-κB antibody (1∶100), the cells were washed twice and incubated with Goat anti-mouse FITC-linked secondary for 1 h. Fluorescence images were visualized using an Olympus Confocal Laser Scanning Biological Microscope and analyzed using Olympus Fluoview Ver. 1.7a viewer software.
3
Immunofluorescence Analysis of Autophagy and Mitophagy Markers
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SLOS and control cells were cultured on cover slips and fixed using 10% formalin buffer. After 30 min of blocking in 3% BSA in PBS, the cover slips were incubated overnight at 4 °C with primary antibodies for LC3B & actin at 1:200 or primary antibodies for Parkin or Pink-1 (Abcam, Cambridge, MA) at 1:300 overnight at 4 °C. Cells were washed three times in PBS and incubated with corresponding secondary antibodies (Cy3 or FITC conjugated anti-rabbit or anti-mouse antibodies) at 1:400 for 1 h. Cells were washed again with PBS and mounted in a medium containing 4′-6-diamidino-2-phenylindole (DAPI) to stain the nuclei (Vector Laboratory, Burlington, Ontario, Canada). Slides were viewed using a Confocal Laser Scanning Biological Microscope (Olympus, Center Valley, PA). Images were captured using Fluoview FV1000 software. A negative control to confirm the specificity of the immunostaining was prepared using non-immune goat serum in place of the primary antibody.
4
Quantification of DNA Damage in FACS-Isolated Cells
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FACS-isolated cells were fixed in PBS/4% paraformaldehyde (PFA) (Sigma-Aldrich Japan) for 10 min at room temperature, then washed in PBS, and smeared on gelatin-coated slides. After air-drying, the slides were rinsed with water to remove salt crystals. They were then permeabilized in PBS/0.2% Triton X-100 (Sigma-Aldrich Japan) for 20 min and blocked in PBS/1% bovine serum albumin (BSA) for 1 h at room temperature. Following this, the slides were incubated in PBS/1% BSA with rabbit H2A.X (phospho S139) antibody (Abcam, Cambridge, MA, USA; cat No.# ab81299) overnight at 4 °C. Slides were then washed five times with PBS (5 min each wash) and incubated for 1 h at 37 °C in PBS/1% BSA with Goat anti-Rabbit IgG (Alexa Fluor 488) (Thermo Fisher Scientific; cat. No. A-11034). Slides were again washed five times with PBS, for a total time of 5 min per wash. Slides were finally incubated with 4′,6-diamidino-2-phenylindole (DAPI) and mounted using Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich Japan; cat. No. F4680). Cells were imaged on a confocal laser scanning biological microscope (Olympus, Tokyo, Japan; cat. No. FV1200 BX61), and positive and negative H2A.X stained cells were also counted, before being classified into three classes: negative (nucleus negatively stained), grade I (H2A.X foci staining part of the nucleus), and grade II (H2A.X foci all over the nucleus).
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