Immunofluorescence and confocal microscopy were conducted as reported [55 (link)] with adjustments. HEK293 cells and HEK293-APE1-R301K KI cells were used in this assay. The pCDNA6.1-APE1-R301F plasmid was transfected into R301K KI cells and labeled R301F group. Empty vectors were transfected into HEK293 cells and R301K KI cells, labeled APE1-WT and APE1-R301K group, respectively. Cells were grown on glass coverslips for 24 h to the desired confluency. The cells were treated with 200 μM H2O2 for 1 h and allowed cells to recover for 2 h before fixation. The cells were gently rinsed with 1×PBS and fixed with paraformaldehyde (3.7%) for 20 min at room temperature. After blocking with 3% fetal bovine serum for 1 h, primary antibodies against APE1, COX Ⅳ were incubated at 4 °C overnight and Alexa 488/568 (Invitrogen) secondary antibodies were used to detect. Cells were mounted in anti-fade solution with DAPI and examined under Zeiss LMS 880 using Airyscan confocal channel with 40×oil objective. Images were collected and processed using Zeiss blue software and sized in Adobe Photoshop CS5.0. The co-localization signal (green + red) beyond nucleus area was determined with Adobe Photoshop by measuring the fluorescence normalized to the number of cell count.
Lms 880
Manufactured by Zeiss
Sourced in Germany
The LMS 880 is a laser scanning microscope manufactured by Zeiss. It is a versatile tool designed for high-resolution imaging and analysis of biological and materials science samples. The LMS 880 utilizes laser technology to capture detailed images, providing researchers with a powerful analytical instrument for their investigations.
Automatically generated - may contain errors
Lab products found in correlation
Mouse on mouse blocking reagent, by Vector Laboratories (2 mentions)
Dapi containing medium, by Vector Laboratories (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti progerin antibody, by Merck Group (2 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Liperfluo, by Dojindo Laboratories (1 mentions)
Liperfluo, by Zeiss (1 mentions)
Liperfluo, by BD (1 mentions)
Bx63 microscope, by Olympus (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Ab109364, by Abcam (1 mentions)
Ab150133, by Abcam (1 mentions)
Ab66047, by Abcam (1 mentions)
Mab356, by Merck Group (1 mentions)
Anti fade mounting medium containing dapi, by Vector Laboratories (1 mentions)
Ab84817, by Abcam (1 mentions)
Ab84818, by Abcam (1 mentions)
Ab229340, by Abcam (1 mentions)
15 protocols using lms 880
1
Immunofluorescence Analysis of APE1 Localization
2
Immunohistostaining of Aortic Lamins
3
Plasmid Construction and Subcellular Localization
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Primers used for plasmid construction are shown in Additional file 6 . Full-length transcripts of CeTIP1;1 and − 2;1 were first isolated using CeTIP1;1F/R and CeTIP2;1F/R as primers, respectively. Then, the PCR products were employed as template, and the CDS without the termination codon were cloned to pNC-Cam1304-SubC, pNC-BiFC-Ecn, and pNC-BiFC-Enn using CeTIP1;1HF/R and CeTIP2;1HF/R as primers. The resulted recombinant plasmids, i.e., pNC-Cam1304-CeTIP1;1, pNC-Cam1304-CeTIP2;1, pNC-BiFC-Ecn-CeTIP1;1, pNC-BiFC-Ecn-CeTIP2;1, pNC-BiFC-Enn-CeTIP1;1, and pNC-BiFC-Ecn-CeTIP2;1 were introduced into Agrobacterium tumefaciens GV3101 with the helper plasmid pSoup-P19 as described before [72 ]. A. tumefaciens-mediated transformation was carried out as previously described [28 (link), 72 ], where tobacco leaves of about 4-week-old plants were used as the receptor. About 48 h after infiltration, transformed leaves were processed for confocal laser scanning microscopy imaging (Zeiss LMS880, Germany). For subcellular localization analysis, markers for tonoplast (AtTIP1;1-RFP [10 (link)]) and plasma membrane (HbPIP2;3-RFP [35 ]) were also co-transformed.
4
Thymocyte Isolation and Analysis
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To maintain the natural attachment between adjacent thymocytes, we gently cut the thymus with surgical scissors in an Eppendorf tube; 1 mL of 1× PBS was added to resuspend the thymocytes, and the thymic debris was allowed to settle. The suspension was pipetted and passed through a 40-μm nylon mesh filter. After counting the cells, approximately 1 × 107 thymocytes were labeled with fluorescent anti-mouse antibodies at 4 °C for 30 min in approximately 100 μL 1× PBS. Then, 30 μL of 4% PFA was directly added and gently mixed, and the cells were fixed at 4 °C for 10 min. The cells were briefly centrifuged at 100×g for 1 min to avoid the formation of a tight cell pellet and resuspended in 200 μL of 1× perm/wash buffer. After staining with 488-labeled phalloidin for 20 min at room temperature, the cells were centrifuged at 100×g for 1 min and resuspended in 60 μL of 1× PBS. For ImageStream experiments, cells were directly examined using an Amnis ImageStream Mk II Imaging Flow Cytometer (Luminex). For immunofluorescence experiments, cells were diluted to a proper concentration, seeded on poly-l -lysine-coated slides and observed by confocal microscopy (ZEISS LMS 880).
5
Quantifying Retinal Ganglion Cell Morphology
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SMI‐32+ RGCs were imaged using confocal microscopy (LMS880, Zeiss, Germany). Z‐stacks images of the whole morphology of individual RGC were acquired and then combined into a single maximum brightness projection image using ImageJ (National Institutes of Health, Bethesda, MD). The Simple Neurite Tracer Image J plug‐in was used to manually trace the dendritic arbor of each cell, and the rendered paths images were generated as previously described (Iaboni et al., 2020 (link)). Parameters were measured as follows—(1) soma area: area of cell body; (2) diameter of soma: the longest diameter of cell body; (3) dendritic field area: the area made from joining the ends of each terminal dendritic branch together using straight lines; (4) arbor asymmetry: the distance between the center of mass of dendritic density and the cell body position; (5) total branches: total number of branches; (6) total dendritic length: the sum of the length of all branches. The soma area, diameter of soma, and dendritic field were calculated using the ImageJ “measure” function. Asymmetry, number of branches, and total dendritic length were calculated based on the rendered paths image. Sholl analysis was performed on the rendered paths image generated above with the Sholl analysis ImageJ plug‐in as described before (Ferreira et al., 2014 (link)).
6
Lysosome Imaging in Fly Thorax
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The experiments were carried out as previously described with some modifications (Macchi et al., 2013 (link)). Fly thoraxes were dissected into halves in Schneider's medium containing 1% cyclodextrin and stained with 1 μM LysoTracker Red DND-99 (Thermo Fisher Scientific) for 5 min. After washing with Schneider's medium for 5 min two times, the specimens were mounted in Schneider's medium and imaged by confocal microscopy (LMS880, Zeiss). Triplicates of volumes of 84.2×84.2×5 μm3 were analyzed. The red fluorescent signals from LysoTracker were measured, and the corresponding volumes were calculated using Imaris image analysis software (Bitplane).
7
Visualizing Lipid Peroxide in Live Cells
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Live cell staining with Liperfluo (Dojindo, Kumamoto, Japan; #L248) was performed according to the manufacturer’s protocols. Briefly, these dyes were diluted in PBS to 1 μM, added to the cell pellets, and incubated in the dark at 37 °C for 10–20 min. The supernatant was removed by centrifugation and the cells were washed three times with PBS. The stained cells were then resuspended in an appropriate volume of PBS and seeded on 4-well glass bottom dishes (Φ9.5 mm/well, MATSUNAMI, Bellingham, WA USA; #D141400). Using a confocal laser scanning microscope (LMS880, Carl Zeiss, Oberkochen, Germany), Liperfluo-stained cells were visualized by acquiring lipid peroxide-derived fluorescence spectra (520–550 nm) in the lambda mode and merging them with the transmitted light image to visualize their localization. Further, the positivity and cell diameter of Liperfluo-stained cells were quantified by BD FACSVerse™ Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For HE staining, cell suspensions were applied to coated glass slides, allowed to stand for 30 min, immediately fixed in 99% ethanol for at least 5 min, washed with water, and stained with H&E. Images were visualized under a BX63 microscope (40× magnification; Olympus, Tokyo, Japan) and calculated using ImageJ software.
8
Immunohistochemical Analysis of Mitochondria and Autophagic Proteins in Fly Thoraxes
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The experiments were carried out as previously described with some modifications (Macchi et al., 2013 (link)). Fly thoraxes were dissected into halves in fixation buffer containing 4% paraformaldehyde and 1% Triton X-100 in PBS and fixed for 20 min at room temperature (RT) without shaking. The specimens were washed with 0.1% Triton X-100 in PBS for 20 min at RT three times. After blocking with 5% normal goat serum (Jackson ImmunoResearch) in 0.1% Triton X-100 in PBS for 2 h at RT, the specimens were stained with primary mouse anti-ATP5A (1:1000, Abcam 14748) and/or primary mouse anti-GABARAP+GABARAPL1+GABARAPL2 antibody (1:500, Abcam ab109364) in blocking buffer overnight at 4°C. After washing with 0.1% Triton X-100 in PBS for 20 min at RT three times, the specimens were stained with secondary antibody (1: 1000 dilutions, anti-mouse IgG Alexa-488, Jackson ImmunoResearch, or Alexa Fluor 647 Phalloidin, Invitrogen A22287) in blocking buffer overnight at 4°C. After washing with 0.1% Triton X-100 in PBS for 20 min at RT three times, the specimens were mounted on the glass slides for imaging. Volumes of 84.2×84.2×5 μm3 were imaged by confocal using LMS880, Zeiss.
9
Immunofluorescence Analysis of APE1 Localization
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Immunofluorescence and confocal microscopy were conducted as reported [55 (link)] with adjustments. HEK293 cells and HEK293-APE1-R301K KI cells were used in this assay. The pCDNA6.1-APE1-R301F plasmid was transfected into R301K KI cells and labeled R301F group. Empty vectors were transfected into HEK293 cells and R301K KI cells, labeled APE1-WT and APE1-R301K group, respectively. Cells were grown on glass coverslips for 24 h to the desired confluency. The cells were treated with 200 μM H2O2 for 1 h and allowed cells to recover for 2 h before fixation. The cells were gently rinsed with 1×PBS and fixed with paraformaldehyde (3.7%) for 20 min at room temperature. After blocking with 3% fetal bovine serum for 1 h, primary antibodies against APE1, COX Ⅳ were incubated at 4 °C overnight and Alexa 488/568 (Invitrogen) secondary antibodies were used to detect. Cells were mounted in anti-fade solution with DAPI and examined under Zeiss LMS 880 using Airyscan confocal channel with 40×oil objective. Images were collected and processed using Zeiss blue software and sized in Adobe Photoshop CS5.0. The co-localization signal (green + red) beyond nucleus area was determined with Adobe Photoshop by measuring the fluorescence normalized to the number of cell count.
10
Mitochondrial Membrane Potential Imaging
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The experiments were carried out as previously described with some modifications (Macchi et al., 2013 (link)). Fly thoraxes were dissected into halves in Schneider's medium containing 1% cyclodextrin and stained with 8 μM JC-1 (Thermo Fisher Scientific) for 30 min. After washing with Schneider's medium for 5 min two times, the specimens were mounted in Schneider's medium and imaged by confocal microscopy (LMS880, Zeiss). Triplicates of volumes of 84.2×84.2×5 μm3 were analyzed. The red and green fluorescent signals from JC1 were measured, and the ratios were calculated using Imaris image analysis software (Bitplane).
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