hTERT-RPE1 WT and WDFY2(–/–) cells were transiently transfected with GFP-VAMP3 and mCherry-CORTACTIN overnight and fixed in 3% PFA. Cells were labeled with anti-GFP (Roche) and RFP-booster (Chromotek) Images were acquired on LSM710 confocal microscope using a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss) objective. The leading edges or cells were selected in the red channel (CORTACTIN). Images were processed in Fiji using a custom Python script, a schematic of the image processing is shown in Supplementary Fig. 9 a . For the generation of superpixels to visualize Vamp3 distribution, cells were outlined using ImageJ and an ImageJ macro (https://gist.github.com/mutterer/035ade419bf9c96475ce ) was used to generate hexagonal ROIs. Mean intensity values for each hexagon were measured and the corresponding hexagon filled with the mean value (Supplementary Fig. 9 b ).
Plan apochromat 63 1.40 oil dic 3
Manufactured by Zeiss
The Plan-Apochromat 63x/1.40 oil DIC III is a high-performance objective lens designed for microscopy applications. It features a numerical aperture of 1.40 and a working distance of 0.19 mm. The lens is optimized for plan-apochromatic correction, providing excellent image quality and flatness of field.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Ar laser multiline, by Zeiss (3 mentions)
Zen 2010, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Rfp booster, by Proteintech (1 mentions)
Anti gfp, by Roche (1 mentions)
Dpss 561, by Zeiss (1 mentions)
Illustrator cs4, by Adobe (1 mentions)
Dpss 561 10, by Zeiss (1 mentions)
Imaris 7, by Oxford Instruments (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Zen black software, by Zeiss (1 mentions)
Orca r2 ccd camera, by Hamamatsu Photonics (1 mentions)
6 protocols using plan apochromat 63 1.40 oil dic 3
1
Quantifying VAMP3 Distribution in WT and WDFY2(-/-) Cells
2
Confocal Microscopy Imaging Workflow
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Confocal images were obtained using LSM710 confocal microscope (Carl Zeiss) equipped with an Ar-laser multiline (458/488/514 nm), a DPSS-561 10 (561 nm), a laser diode 405–30 CW (405 nm), and a HeNe laser (633 nm). The objective used was a Plan-Apochromat 63 × /1.40 oil DIC III (Carl Zeiss). Image processing was performed with basic software ZEN 2010 (Carl Zeiss) and Fiji software.
3
VAMP3 Vesicle Distribution in WT and WDFY2-KO Cells
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hTERT-RPE1 WT and WDFY2(−/−) cells were transfected with GFP-VAMP3 and fixed in 3% PFA. Cells were labeled with human anti-EEA1 antibody and rabbit anti-Lamp1 antibody and corresponding fluorophore-conjugated secondary antibodies. Images were acquired using a LSM710 confocal microscope using a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss) objective. To measure VAMP3 distribution, EEA1 and LAMP1-positive vesicles were automatically segmented and GFP-VAMP3 intensity was measured within the respective compartments using a custom Python-based Fiji script26 (link). Individual measurements were then collected and plotted.
4
Confocal Imaging Using Zeiss Microscopes
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Confocal images were obtained using LSM710 or LSM780 confocal microscope (Carl Zeiss) equipped with an Ar-laser multiline (458/488/514 nm), a DPSS-561 10 (561 nm), a laser diode 405-30 CW (405 nm), and an HeNe laser (633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss). ZEN 2010 (Carl Zeiss), Photoshop CS4 (Adobe), and ImageJ software were used for brightness and contrast adjustments and for making figures and insets. Imaris 7.1.1 (Bitplane AG) was used for 3D rendering of confocal z-stacks. Illustrator CS4 (Adobe) was used for making figures and insets.
5
Confocal Microscopy Imaging Technique
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Confocal images were obtained using LSM710 confocal microscope (Carl Zeiss) equipped with an Ar-laser multiline (458/488/514 nm), a DPSS-561 10 (561 nm), a laser diode 405-30 CW (405 nm), and a HeNe laser (633 nm). Images were taken using a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss).
6
Epifluorescent and Confocal Imaging Protocol
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Epifluorescent images were obtained using an IX83 Olympus microscope equipped with a Hamamatsu Orca R2 CCD camera. The objective used was a PLAPON 60×/1.42 Oil (Olympus). The Pearson correlation coefficient and the area of the cell were measured in FIJI ImageJ [16 (link)] software.
Confocal images were obtained using an LSM880 confocal microscope (Carl Zeiss) equipped with an Argon-laser multiline (458/488/514 nm) and HeNe laser (543 nm and 633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss). For ultrastructure visualization, the airy scan mode was used. Total fluorescence intensities (TFI) and the area of cells were measured using ZEN black software (Carl Zeiss), quantified on maximum z-projections, and expressed over the area (size) of cells. For fluorescence intensity measurements, imaging conditions between different samples were kept constant. Fluorescence intensity was always expressed over the area (size) of cells. Colocalization studies were performed using ZEN black software (Carl Zeiss).
Confocal images were obtained using an LSM880 confocal microscope (Carl Zeiss) equipped with an Argon-laser multiline (458/488/514 nm) and HeNe laser (543 nm and 633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss). For ultrastructure visualization, the airy scan mode was used. Total fluorescence intensities (TFI) and the area of cells were measured using ZEN black software (Carl Zeiss), quantified on maximum z-projections, and expressed over the area (size) of cells. For fluorescence intensity measurements, imaging conditions between different samples were kept constant. Fluorescence intensity was always expressed over the area (size) of cells. Colocalization studies were performed using ZEN black software (Carl Zeiss).
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