HaloTag-LC3 assays were performed as previously described (Takahashi et al., 2018 (link)). Briefly, cells were transfected with HaloTag-LC3 for 24 h and treated with 20 μM digitonin in KHM buffer at 37°C for 2 min. Then cells were incubated with membrane-impermeable ligand (MIL) HaloTag® Alexa Fluor® 488 ligand (G1001, Promega) at 37°C for 15 min. Cells were fixed in 4% PFA for 20 min at room temperature, washed 3 times with PBS, and stained with membrane-permeable ligand (MPL) HaloTag® TMR ligand (G8251, Promega) for 30 min at room temperature. Coverslips were mounted on microscope slides with DAPI in 50% glycerol and examined by a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss).
Axiovert zoom
Manufactured by Zeiss
The Zeiss Axiovert zoom is a versatile microscope system designed for a wide range of laboratory applications. It features a zoom optical system that allows for continuous magnification adjustment, providing users with the flexibility to observe samples at various magnifications. The Axiovert zoom is a reliable and precise tool for scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 meta, by Zeiss (8 mentions)
Axiocam hrm, by Zeiss (4 mentions)
Plan apochromat, by Zeiss (3 mentions)
Digitonin, by Merck Group (2 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (2 mentions)
Halotag tmr ligand, by Promega (1 mentions)
Halotag alexa fluor 488 ligand, by Promega (1 mentions)
Plan apochromatlan, by Zeiss (1 mentions)
Prism 8, by GraphPad (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Digitonin d141, by Merck Group (1 mentions)
9 protocols using axiovert zoom
1
Monitoring Autophagy Flux Using HaloTag-LC3
2
Immunostaining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on coverslips for immunostaining. After washing 3 times with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), cells were fixed with 4% PFA for 15 min at room temperature, and then permeabilized in cold digitonin (Sigma, D141) for 10 min at 4°C. After blocking with 5% goat serum for 60 min at room temperature, cells were incubated with primary antibodies (diluted in 5% goat serum) overnight at 4°C. Then cells were washed 3 times with PBS and stained with fluorescently-labeled secondary antibodies for 60 min at room temperature. Lysosomes were stained with LysoTracker™ Deep Red (1:1000, L12492, Thermo Fisher Scientific) for 30 min at 37°C and 5% CO2. Coverslips were mounted on microscope slides with DAPI in 50% glycerol. Images were acquired with a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss) with a 63×/1.40 oil-immersion objective lens (Plan-Apochromatlan, Zeiss) and a camera (Axiocam HRm, Zeiss).
3
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured on coverslips in cell culture plates. After harvesting, cells were washed with 1×PBS and fixed with 4% PFA for 20 min at room temperature and then permeabilized by 10 mg/ml digitonin (D141; Sigma) for 10 min. Cells were blocked by 5% goat serum for 1 h and incubated with primary antibodies (diluted by 5% goat serum) at 4°C overnight. After washing three times with 1×PBS (10 min for each), cells were incubated with fluorescently conjugated secondary antibodies (diluted by 5% goat serum) for 1 h at room temperature. After three washes with 1×PBS, coverslips were placed on microscope slides with DAPI in 50% glycerol. LysoTracker Deep Red (1:1,000, L12492; Thermo Fisher Scientific) was used to stain lysosomes for 30 min at 37°C and 5% CO2. Cells were examined with a confocal microscope (LSM 880 Meta plus Zeiss Axiovert Zoom; Zeiss) equipped with a 63×/1.40 oil immersion objective lens (Plan-Apochromat; Zeiss) and a camera (AxioCam HRm; Zeiss).
4
FRAP Analysis of Liquid Droplet Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence recovery after photobleaching (FRAP) was studied in liquid droplets containing mCherry-tagged proteins by confocal microscopy (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss) at room temperature. ROI was first photobleached by excitation at 405 nm. The fluorescence intensity of the photobleached area was then collected every 2 s. Image intensity was measured by Mean ROI in the Zeiss Zen software during the acquisition time, and graphs drawn by Prism8 (GraphPad).
5
Digitonin-Mediated Proteinase K Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated on glass bottom dishes (801001, NEST) and transfected with the indicated plasmids for 24 h. After washing with KHM buffer (100 mM potassium acetate, 20 mM HEPES, 2 mM MgCl2, pH 7.4), cells were incubated with 20 μM digitonin for 5 min. Then cells were treated with 50 μg/ml proteinase K (P2308, Sigma) and real-time images were immediately recorded by a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss).
6
Protein Concentration Quantification by Fluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Only one component was labeled with Sulfo-Cyanine3 maleimide (red). To generate a standard calibration curve, the fluorescence intensity of a protein at different concentrations was measured by confocal microscopy (LSM 880 Meta plus Zeiss Axiovert zoom; Zeiss) and analyzed by the ImageJ software. The protein concentration in the measured droplets was obtained by fitting the measured fluorescence intensity into the standard calibration curve.
7
Fluorescence Recovery After Photobleaching
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FRAP was performed with a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom; Zeiss) at room temperature. Fluorescence-labeled protein was adjusted to 1% by diluting the labeled protein into the unlabeled one. Defined regions were photobleached at a specific wavelength using the 561-nm or 488-nm laser, and the fluorescence intensities in these regions were collected every 3 s (for in vitro droplets) and normalized to the initial intensity before bleaching. Image intensity was measured by mean mean region of interest and further analyzed by Prism (GraphPad).
8
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured on coverslips were washed three times with PBS, fixed with 4% PFA for 20 min, and permeabilized for 10 min at RT with 100 μg/ml Digitonin (D141; Sigma-Aldrich) for permeabilization of the plasma membrane or 5‰ Triton X-100 for permeabilization of the nuclear membrane. Cells were blocked with 5% goat serum for 60 min and then incubated with the indicated primary antibodies overnight at 4°C. After three washes with PBS, cells were stained with fluorescently labeled secondary antibodies for 1 h at RT and observed using a confocal microscope (LSM 880 Meta plus Zeiss Axiovert Zoom; Zeiss) equipped with a 63×/1.40 oil-immersion objective lens (Plan-Apochromat; Zeiss) and a camera (AxioCam HRm; Zeiss) at RT.
9
FRAP Analysis of TFEB Protein Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Droplets formed by Cy3-labeled MBP-TFEB protein were used for FRAP analysis. TFEB-GFP puncta in HeLa cells were used for the in vivo FRAP assay. The experiments were performed on a confocal microscope (LSM 880 Meta plus Zeiss Axiovert Zoom; Zeiss) equipped with a 63×/1.40 oil-immersion objective lens (Plan-Apochromat; Zeiss) and a camera (AxioCam HRm; Zeiss) at RT. The in vivo FRAP assays were performed at 37°C with 5% CO2. Selected regions were photobleached at 561 nm for Cy3 and 488 nm for GFP, and the fluorescence intensities in these regions were collected every 2 s as mean region of interest. The value of signals was normalized to the initial intensity before photobleaching. Recovery curves were plotted using GraphPad Prism software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!