Human olfactory cells were fixed in PBS containing 4% PFA for 15 min at room temperature, washed 3X in PBS, and incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 3% horse serum) for 30 min at room temperature. For newborn pigs, olfactory tissues were removed, postfixed overnight in 0.1 M phosphate buffer (PB) containing 4% PFA, and later cryoprotected in 0.1 M PB buffer containing 30% sucrose. Samples were embedded in Tissue-Tek OCT compound, snap-frozen in cold isopentane, and processed on a Leica CM 3050S cryostat. Olfactory epithelia were cut in 16-μm-thick coronal sections and were directly mounted on SuperFrost Plus slides glasses (Thermo Fisher Scientific). Olfactory bulbs were cut in 30-μm serial free-floating sagittal sections in a PBS solution. Sections were treated with 10 mM sodium citrate for 5 min at 95° to 100°C for antigen retrieval, washed (3× 5 min) with PBS, incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 5% normal horse serum) for 2 hours at room temperature (RT), incubated overnight at 4°C in blocking solution supplemented with the primary antibody, washed in PBS solution, and incubated in blocking solution supplemented with secondary antibody for 1 hour at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Sigma-Aldrich) for 5 min. We used the following primary antibodies: goat anti-SOX2 (1:300; R&D Systems, AF2018), rabbit anti-KRT5 (1:800; BioLegend, 905501), goat anti-OMP (1:2000; Wako, 019-22291), mouse anti-NGFR (1:1000; Sigma-Aldrich, N5408), mouse anti-PCNA (1:1000; Sigma-Aldrich, P8825), rabbit anti-DCX (1:2000; Abcam, AB18723), rabbit anti-Ki67 (1:100; Abcam, ab9021), mouse anti-Gαolf (1:500; Santa Cruz Biotechnology, sc-55545), rabbit anti-ITPR3 (1:500; Millipore, AB9076), rabbit anti-TRPM5 (1:400; Alomone Labs, ACC-045), and rat anti-NES (1:600; Santa Cruz Biotechnology, sc-33677). Secondary antibodies used for the corresponding target species were conjugated with Alexa Fluor 488, Alexa Fluor 546, and Alexa Fluor 647 (1:500; Thermo Fisher Scientific). Epithelium thickness, cell density, areas, and OB glomeruli quantifications were preformed from images acquired on a Zeiss LSM-780 confocal laser-scanning microscope. Image regions were analyzed in the entire z axis with 3-μm step intervals, and images were reconstituted using the maximum intensity projection tool of Zen software. Images were analyzed with Fiji/ImageJ (NIH). Demarcations of epithelium limits and cell-specific layers were based on OMP and NGFR immunoreactivities, and DAPI+ cells were counted using the particle analyzer plug-in of Fiji. Counts of the number of cells were evaluated blindly for each animal. Five slices per animal and genotype were used. Cells were counted from five to seven independent animals. For each sample, images were acquired at least from five different anatomical levels. Olfactory bulb glomeruli demarcation was based on OMP immunoreactivity in 5 to 17 slices per animal from six independent animals per genotype. A total of 819 glomeruli (469 CFTR+/+ and 350 CFTR−/−) covering all main OB topography (dorsal, ventral, lateral, and medial) were analyzed.
4 6 diamidino 2 phenylindole (dapi)
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DAPI is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used as a nuclear counterstain in fluorescence microscopy to visualize and locate cell nuclei.
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4',6-Diamidino-2-phenylindole (DAPI) is an actively commercialized product by the Merck Group under its Sigma-Aldrich brand. The product is available for purchase through the official Sigma-Aldrich website.
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22 679 protocols using «4 6 diamidino 2 phenylindole (dapi)»
1
Immunofluorescence Analysis of Olfactory Tissues
2
Immunofluorescence Analysis of Hippocampal Neurogenesis
For
immunofluorescence experiments, three brain sagittal sections between
0.60 and 1.20 mm (approximately) lateral to the midline in Paxinos
and Franklin’s Mouse Brain Atlas44 were used. Free-floating serial sections (50 μm thick) were
first rinsed in PB and then preincubated for 2 h in PB with 0.25%
Triton-X100 and 3% normal serum of the species in which the secondary
antibodies were raised (Normal Donkey Serum, Invitrogen, Thermo Scientific).
Subsequently, brain sections were incubated for 24h at 4 °C in
the same preincubation stock solution (PB + Triton + Serum) containing
different combinations of the following primary antibodies: L1516
(Merck) for Lectin from Wisteria floribunda (WFA) and Aggrecan (Merck,
ab1031). After rinsing in the sections in PB, they were incubated
for 2 h at room temperature in anti-streptavidin Alexa 488 (Thermo
Fisher, S-32254) and donkey antirabbit 267 Alexa 594 (Thermo Fisher,
A-21207). Sections were also labeled with the nuclear stain DAPI (4,6-diamidino2-phenylindole;
Sigma, St. Louis, MO). We obtained stitched image stacks from the
entire DG in the hippocampus. These were recorded at 1-μm intervals
through separate channels with a 20× lens for the analysis of
neurogenesis and the extracellular matrix (ECM). The same range of
z-slices was obtained from each slide in each experiment. Adobe Photoshop
(CS4) software was used to build the figures.
Immunohistochemical
quantifications for perineuronal nets (PNN) of the ECM, were performed
by counting positive units labeled with WFA in the DG (including the
GCL and hilus) from three consecutive medial brain sections. To determine
the density of different cells, the different layers of DG were traced
on the DAPI channel of the z projection of each confocal stack of
images, and the area of this structure was measured using the freehand
drawing tool in Fiji. This area was multiplied by the stack thickness
to calculate the reference volume. The number of positive cells was
divided by the reference volume, and the density (number of cells/mm3)
of cells was calculated.
immunofluorescence experiments, three brain sagittal sections between
0.60 and 1.20 mm (approximately) lateral to the midline in Paxinos
and Franklin’s Mouse Brain Atlas44 were used. Free-floating serial sections (50 μm thick) were
first rinsed in PB and then preincubated for 2 h in PB with 0.25%
Triton-X100 and 3% normal serum of the species in which the secondary
antibodies were raised (Normal Donkey Serum, Invitrogen, Thermo Scientific).
Subsequently, brain sections were incubated for 24h at 4 °C in
the same preincubation stock solution (PB + Triton + Serum) containing
different combinations of the following primary antibodies: L1516
(Merck) for Lectin from Wisteria floribunda (WFA) and Aggrecan (Merck,
ab1031). After rinsing in the sections in PB, they were incubated
for 2 h at room temperature in anti-streptavidin Alexa 488 (Thermo
Fisher, S-32254) and donkey antirabbit 267 Alexa 594 (Thermo Fisher,
A-21207). Sections were also labeled with the nuclear stain DAPI (4,6-diamidino2-phenylindole;
Sigma, St. Louis, MO). We obtained stitched image stacks from the
entire DG in the hippocampus. These were recorded at 1-μm intervals
through separate channels with a 20× lens for the analysis of
neurogenesis and the extracellular matrix (ECM). The same range of
z-slices was obtained from each slide in each experiment. Adobe Photoshop
(CS4) software was used to build the figures.
Immunohistochemical
quantifications for perineuronal nets (PNN) of the ECM, were performed
by counting positive units labeled with WFA in the DG (including the
GCL and hilus) from three consecutive medial brain sections. To determine
the density of different cells, the different layers of DG were traced
on the DAPI channel of the z projection of each confocal stack of
images, and the area of this structure was measured using the freehand
drawing tool in Fiji. This area was multiplied by the stack thickness
to calculate the reference volume. The number of positive cells was
divided by the reference volume, and the density (number of cells/mm3)
of cells was calculated.
3
Oxidative Stress Assessment in Chondrocytes
After the treatments, primary condylar chondrocytes were incubated with 5 µmol/L of BODIPY581/591 C11 (Invitrogen, D3861) for 30 min, trypsinized and filtered into single cell suspensions. Flow cytometry analysis (Becton Dickinson) was performed using the FITC filter for oxidized BODIPY-C11 (emission: 510 nm) and PE-TexasRed filter for reduced BODIPY-C11 (emission: 590 nm). About 20 000 cells were analyzed for each sample. FlowJo v10 (BD Bioscience) was used for data analysis. After the treatments, primary condylar chondrocytes were stained with DCFH-DA (Beyotime, S0033) to measure ROS and assayed by fluorescence microscopy (Ts2R/FL; Nikon). Fixed chondrocytes were stained with 0.1 µg/mL Nile Red (MCE, HY-D0718) for 30 minutes, and the nuclei were counterstained using DAPI (Sigma). Images were captured with a Nikon A1 confocal microscope and analyzed with ImageJ software.
4
Multi-Dimensional Immune Cell Profiling from Murine Skin
Single-cell suspensions were prepared from murine back skin as described above or from organoid cultures by mechanical homogenization and incubation in 0.5% trypsin (Gibco) and 0.5 mM EDTA for 10 min at 37°C. Cells were rinsed once with KGM and stained with fluorescently labeled antibodies for 30 min on ice. After two washes with FACS Buffer (2% fetal calf serum, 2 mM EDTA in PBS), cells were analyzed in a BD FACSCanto II or sorted in either a BD FACSAria II or a BD FACSAria Fusion. Data were analyzed using FlowJo software version 10.9. Expression of cell surface markers was analyzed on live cells after exclusion of cell doublets and dead cells using 7AAD (eBioscience), DAPI (Sigma-Aldrich), or fixable viability dye 405 (eBiosciences). Apoptotic cells were labeled with Alexa Fluor 555–Annexin V (Invitrogen). The following antibodies were used: eFluor660-or FITC-CD34 (clone RAM34; eBioscience), and PE-Cy7-or FITC-α6 integrin (clone GoH3; eBioscience).
Immune cell FACS was performed from ear skin from which cells were isolated using DNAse I 40 µg/ml (11284932001; Roche) and 20 U/ml Collagenase Type I Worthington (LS0004194) for 90 min at 37°C in RPMI1640, followed by gentleMACS tissue dissociation using gentleMACS C tubes and subsequent separation using 70-µm cell strainers. After centrifugation at 300 g, 4°C, for 10 min, cells were resuspended in 500 μl PBS containing 2% FBS.
The following antibodies were used: Fc Block (14-0161-86; Invitrogen), live/dead eF780 (65-0865-14; Invitrogen), CD45 FITC (103108; BioLegend), Gata3 PE (12-9966-42; eBioscience), NKp46 eF710 (46-3351-82; eBioscience), CD4 PE-Cy7 (100421; BioLegend), Eomes eF660 (50-4875-82; eBioscience), CD90.2 AF700 (105320; BioLegend), Rorγt BV421 (562894; BD Biosciences), CD25 BV 605 (563061; BD Biosciences), CD11b super bright 702 (67-0112-82; eBioscience), CD3 biotin (344820; BioLegend), CD19 biotin (115504; BioLegend), TCRb biotin (109204; BioLegend), TCRgd biotin (13-5711-82; eBioscience), CD5 biotin (BioLegend [100604], FCeR1 biotin [134304; BioLegend], and SA V510 anti-biotin [405234; BioLegend]).
Immune cell FACS was performed from ear skin from which cells were isolated using DNAse I 40 µg/ml (11284932001; Roche) and 20 U/ml Collagenase Type I Worthington (LS0004194) for 90 min at 37°C in RPMI1640, followed by gentleMACS tissue dissociation using gentleMACS C tubes and subsequent separation using 70-µm cell strainers. After centrifugation at 300 g, 4°C, for 10 min, cells were resuspended in 500 μl PBS containing 2% FBS.
The following antibodies were used: Fc Block (14-0161-86; Invitrogen), live/dead eF780 (65-0865-14; Invitrogen), CD45 FITC (103108; BioLegend), Gata3 PE (12-9966-42; eBioscience), NKp46 eF710 (46-3351-82; eBioscience), CD4 PE-Cy7 (100421; BioLegend), Eomes eF660 (50-4875-82; eBioscience), CD90.2 AF700 (105320; BioLegend), Rorγt BV421 (562894; BD Biosciences), CD25 BV 605 (563061; BD Biosciences), CD11b super bright 702 (67-0112-82; eBioscience), CD3 biotin (344820; BioLegend), CD19 biotin (115504; BioLegend), TCRb biotin (109204; BioLegend), TCRgd biotin (13-5711-82; eBioscience), CD5 biotin (BioLegend [100604], FCeR1 biotin [134304; BioLegend], and SA V510 anti-biotin [405234; BioLegend]).
5
Detailed Methodology for Cell-Based Assays
In this study, we utilized high-purity chemicals from various suppliers. Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Cat. #D5648) was supplemented with sodium bicarbonate (Sigma-Aldrich), fetal bovine serum (FBS, Gibco), penicillin (Gibco), streptomycin (Gibco), sodium pyruvate (Gibco), and amphotericin B (Gibco). Sparstolonin B (SsnB, ≥98%) was purchased from Merck Millipore (Cat. #SML1767) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, ≥99.9%). Phorbol 12-Myristate 13-Acetate (PMA, ≥98%) was obtained from LC Laboratories (Cat. #P-1680) and dissolved in DMSO (Sigma-Aldrich, ≥99.9%). MTT (≥99%) was from Gold Biotechnology and dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich). Immunofluorescence reagents included paraformaldehyde, Triton X-100 (Sigma-Aldrich), normal goat serum (Vector Laboratories), and primary antibodies from Bioss Antibodies, Affinity Biosciences, BT Lab, and Cell Signaling Tech., with secondary antibody Alexa Fluor (Abcam) and DAPI (Sigma-Aldrich). The ELISA kits were purchased from BT Lab and Biotechnology. The TUNEL assay kit was purchased from Elabscience. Flow cytometry reagents included Annexin-V and propidium iodide (Elabscience). Sphingolipidomic reagents included chloroform, methanol, ammonium formate, and formic acid (Sigma-Aldrich). Standards were purchased from Avanti Polar Lipids, and the internal standard was purchased from Cambridge Isotope Laboratories. LC-MS/MS analysis involved the use of the LCMS-8040 system (Shimadzu) with an XTerra C18 column (Waters).
Top 5 most cited protocols using «4 6 diamidino 2 phenylindole (dapi)»
1
Intracellular Labeling of Cortical Neurons
Four 9 month old male mice (C57Bl/SJL) were used. Animals were anesthetized with choral hydrate (15% aqueous solution, i.p.) and were perfused transcardially with 4% paraformaldehyde and 0.125% glutaraldehyde in phosphate buffer saline (PBS; pH 7.4). The brains were then carefully removed from the skull and postfixed for 6 hours. All procedures were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Mount Sinai School of Medicine Institutional Animal Care and Uses Committee.
For intracellular injections, brains were coronally sectioned at 200 µm on a Vibratome (Leica, Nussloch, Germany). The sections were then incubated in 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA), a fluorescent nucleic acid stain, for 5 minutes, mounted on nitrocellulose filter paper and immersed in PBS. Using DAPI as a staining guide, individual layer II/III pyramidal neurons of the frontal cortex were loaded with 5% Lucifer Yellow (Molecular Probes, Eugene, OR, USA) in distilled water under a DC current of 3–8 nA for 10 minutes, or until the dye had filled distal processes and no further loading was observed [45] (link), [49] (link). Tissue slices were then mounted and coverslipped in Permafluor. Dendritic segment and spine imaging was performed using a Zeiss 410 confocal laser scanning microscope (Zeiss, Thornwood, NY, USA) using a 488 nm excitation wavelength, using a 1.4 N.A. Plan-Apochromat 100× objective with a working distance of 170 µm and a 5× digital zoom. After gain and offset settings were optimized, segments were digitally imaged at 0.1 µm increments, along the optical axis. The confocal stacks were then deconvolved with AutoDeblur (MediaCybernetics, Bethesda, MD, USA).
Supporting Information is available online (Box S1 )
For intracellular injections, brains were coronally sectioned at 200 µm on a Vibratome (Leica, Nussloch, Germany). The sections were then incubated in 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA), a fluorescent nucleic acid stain, for 5 minutes, mounted on nitrocellulose filter paper and immersed in PBS. Using DAPI as a staining guide, individual layer II/III pyramidal neurons of the frontal cortex were loaded with 5% Lucifer Yellow (Molecular Probes, Eugene, OR, USA) in distilled water under a DC current of 3–8 nA for 10 minutes, or until the dye had filled distal processes and no further loading was observed [45] (link), [49] (link). Tissue slices were then mounted and coverslipped in Permafluor. Dendritic segment and spine imaging was performed using a Zeiss 410 confocal laser scanning microscope (Zeiss, Thornwood, NY, USA) using a 488 nm excitation wavelength, using a 1.4 N.A. Plan-Apochromat 100× objective with a working distance of 170 µm and a 5× digital zoom. After gain and offset settings were optimized, segments were digitally imaged at 0.1 µm increments, along the optical axis. The confocal stacks were then deconvolved with AutoDeblur (MediaCybernetics, Bethesda, MD, USA).
Supporting Information is available online (
Corresponding organizations : Imaging Center, Icahn School of Medicine at Mount Sinai
2
Fluorescent Imaging of Golgi Organization
Cells were cultured on glass coverslips (Matsunami) pre-coated with 10 µg/ml fibronectin (Sigma) and fixed with 4% (w/v) paraformaldehyde in PBS or BRB80 [80 mM Pipes (pH 6.8), 1 mM MgCl2, and 1 mM EGTA] for 10 min at room temperature. Fixed cells were stained with the respective antibodies, phalloidin conjugated with either Alexa Fluor 488 or rhodamine (Invitrogen), along with DAPI (Sigma) as described previously2 (link), 54 (link). In situ proximity ligation assay (PLA) was performed using Duolink kit (Olink Bioscience) according to the manufacturer’s instructions. After completion of the PLA reaction, samples were refixed with 4% (w/v) paraformaldehyde and incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) to detect the individual proteins. Fluorescence images were obtained using a laser scanning confocal imaging system (LSM700, Carl Zeiss) and processed using the ImageJ software. Number of Golgi fragments was quantified by using the ImageJ particle analysis tool. Colocalization was examined using the ImageJ JACoP plugin64 (link) or Metamorph (Molecular Devices).
Corresponding organizations : Kobe University, Brigham and Women's Hospital, Harvard University, RIKEN Center for Brain Science, Shimane University, University of Massachusetts Chan Medical School
3
Immunofluorescence Microscopy Techniques
For immunofluorescence, cells cultured on glass coverslips were fixed with either 4% PFA or 100% methanol. After fixation in methanol, cells were progressively rehydrated by successive washes with 85%, 70%, and 50% ethanol in water and two final washes with PBS. PFA-fixed cells were permeabilized with 0.2% Triton X-100. After blocking by incubation with 1% BSA in PBS, the slides were incubated with the primary antibodies, washed, and incubated with species-specific fluorochrome-tagged secondary antibodies. They were washed again and incubated with DAPI (Sigma-Aldrich) to stain the nuclei. The coverslips were mounted in Mowiol supplemented with an anti-fading agent (AF100; Biovalley). Specimens were examined with either an ApoTome imaging system (Imager Z1) equipped with a 63×/1.4 NA oil differential interference contrast (DIC) objective lens or a confocal microscope (LSM700) equipped with a 63× Plan Apochromat/1.4 NA oil objective lens (all from Carl Zeiss). Images were acquired and analyzed with AxioVision or Zen software (Carl Zeiss). For live-cell imaging, cells were cultured in 35-mm-diameter glass-bottomed dishes (MatTek Corporation). Before analysis, Hepes was added to a final concentration of 10 mM. Video microscopy was performed with two spinning-disk confocal microscopes (UltraView VOX and UltraView ERS; PerkinElmer) equipped with a FRAP system (PhotoKinesis). TIRF imaging was done with a CellM system (Olympus) with a Plan Apochromat 100×/1.45 NA objective lens (Olympus). For TIRF experiments, the laser beam was adjusted so that only a thin sheet below the coverslip (∼100 nm) was illuminated. Noninvasive imaging required low laser power compensated by a relatively long exposure time. We achieved a frame rate of 2 images/s. FRAP experiments were performed as follows: a single, optically sectioned plane of the cell was imaged every second for 5 frames, and an area of ∼3–5 µm in size within the cell was then bleached with a laser beam. Fluorescence recovery was then followed, at a frame rate of 1 image/s. Half recovery times and immobile fractions were derived by fitting a double-exponential model, the simplest model that reliably fitted recovery curves.
Corresponding organizations : Centre National de la Recherche Scientifique, Biotechnologie et Signalisation Cellulaire, Institut Pasteur, Rockefeller University, University of Lisbon, MRC Laboratory for Molecular Cell Biology, University College London, Medical Research Council, Council for Scientific and Industrial Research
4
Immunostaining and Quantification of Mitophagy and Autophagy Markers
HeLa cells, seeded in 2-well chamber slides (Lab-Tek), were treated as indicated in the figures legends. Following treatment, cells were rinsed in PBS and fixed for 15 min at RT with 4% paraformaldehyde. Cells were then permeabilized and blocked with 0.1% Triton X-100, 3% goat serum in PBS for 40 minutes at RT. For immunostaining, cells were incubated with antibodies (as indicated in figure legends) diluted in 3% goat blocking serum overnight at 4 °C, then rinsed with PBS and incubated with either anti- rabbit or mouse Alexa Fluor- 488 and 633 conjugated secondary antibodies (Life Technologies), or anti-chicken Alexa Fluor 488 conjugated antibody (Life Technologies) for 1 h at RT. Cells were washed 3 times for 5 min each with 1% Triton X-100, PBS. During the final wash step, cells were incubated with DAPI (10 μg/mL DAPI, Sigma) in PBS for 5 min. To measure mitophagy by mitochondrial DNA (mtDNA) immunostaining; images were collected from samples stained with DAPI and immunostained for DNA using a plan-Apochromat 63×/1.4 oil DIC objective on an LSM 510 microscope (Zeiss). Four image slices were collected through the Z plane encompassing the top and bottom of the cells. Image analysis was performed on all images collected in the Z plane using Volocity software (Perkin Elmer v6.0.1). The percent mtDNA stain remaining was calculated using the following formula: (cDNAv-nDNAv)/n, where cDNAv= the total cellular DNA volume determined by staining using anti-DNA antibodies and, nDNAv = the total nuclear DNA stain volume determined using DAPI, and n= the number of cells. The mtDNA stain volume in untreated cells was normalized to 100% and the amount of mtDNA stain remaining after drug treatment was subsequently determined. Final values represent data acquired from 50–200 cells from three independent experiments.
To analyze LC3/mitochondria protein colocalization; cells were treated, fixed and immunostained as above. Between 5–8 slices were imaged through the Z plane using either a plan-Apochromat 63× or 100×/1.4 oil DIC objective on a CW STED confocal microscope (Leica). Volocity software (Perkin Elmer, v6.0.1) was used to measure intensity of the GFP signal representing LC3 in the volume occupied by mitochondria (as defined by Tom20 positive region) and the cytosol (as defined by Tom20 negative region). “Normalized mitochondrial LC3” was calculated using the following formula: Normalized mitochondrial LC3 = (mi/mv)/(ci/cv), where mi = mitochondrial GFP intensity, mv = mitochondrial volume, ci = cytosolic GFP intensity and cv= cytosolic volume. The resulting Normalized mitochondrial LC3 is equal to 1 if the intensity of GFP is equal per volume in the cytosolic and mitochondrial volumes (no translocation) and is above one if the mitochondrial intensity is higher per volume (translocation). Final values for Normalized mitochondrial LC3 represents data acquired from 50–105 cells from three independent experiments.
For GFP-DFCP1, GFP-WIPI1 and GFP-ULK1 puncta analysis; cells were treated, prepared and imaged on the CW STED as above with the addition of immunofluorescence using either rabbit or chicken GFP antibodies to enhance the signal in the green channel. For GFP-DFCP1, puncta were quantified using Volocity software (Perkin Elmer v6.0.1) and for GFP-WIPI1 and GFP-ULK1 puncta were quantified manually. Colocalization of autophagy receptors with GFP-DFCP1 or GFP-ULK1 was assessed with line scans using LAS AF software (Leica, v.2.6.0.7266).
To analyze LC3/mitochondria protein colocalization; cells were treated, fixed and immunostained as above. Between 5–8 slices were imaged through the Z plane using either a plan-Apochromat 63× or 100×/1.4 oil DIC objective on a CW STED confocal microscope (Leica). Volocity software (Perkin Elmer, v6.0.1) was used to measure intensity of the GFP signal representing LC3 in the volume occupied by mitochondria (as defined by Tom20 positive region) and the cytosol (as defined by Tom20 negative region). “Normalized mitochondrial LC3” was calculated using the following formula: Normalized mitochondrial LC3 = (mi/mv)/(ci/cv), where mi = mitochondrial GFP intensity, mv = mitochondrial volume, ci = cytosolic GFP intensity and cv= cytosolic volume. The resulting Normalized mitochondrial LC3 is equal to 1 if the intensity of GFP is equal per volume in the cytosolic and mitochondrial volumes (no translocation) and is above one if the mitochondrial intensity is higher per volume (translocation). Final values for Normalized mitochondrial LC3 represents data acquired from 50–105 cells from three independent experiments.
For GFP-DFCP1, GFP-WIPI1 and GFP-ULK1 puncta analysis; cells were treated, prepared and imaged on the CW STED as above with the addition of immunofluorescence using either rabbit or chicken GFP antibodies to enhance the signal in the green channel. For GFP-DFCP1, puncta were quantified using Volocity software (Perkin Elmer v6.0.1) and for GFP-WIPI1 and GFP-ULK1 puncta were quantified manually. Colocalization of autophagy receptors with GFP-DFCP1 or GFP-ULK1 was assessed with line scans using LAS AF software (Leica, v.2.6.0.7266).
Corresponding organizations : National Institute of Neurological Disorders and Stroke, National Institutes of Health
5
Isolation and Characterization of Mouse Hematopoietic Stem Cells
+ Expand
Corresponding organizations : Children's Medical Center, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center
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