Immunofluorescence studies were carried out as previously described [24] (link). Briefly, cells grown overnight on 24-well cover glasses (Menzel, 12 mm) were treated with Cy2/Cy3-labeled MOA in FCS-containing medium and fixed in 4% paraformaldehyde (PFA) at 4 °C for 10 min. Cells were washed and incubated with NH4Cl at room temperature for 15 min before being permeabilized with PBS containing 0.02% saponin and 0.2% bovine serum albumin (BSA). Cells were stained with either anti-EEA1 (1:50, BD Transduction Laboratories), anti-Calnexin (1:100, Enzo Life Science), anti-CTR 433 (1:100, kind gift of Michel Bornens, Curie Institut), anti-Giantin (1:100, Abcam), anti-TGN46 (1:100, Sigma Aldrich), anti-Transferrin receptor (TfR, 1:100, Life Technologies), anti-Rab11 (1:100, Cell Signaling), anti-Lamp1 (1:200, BD PharMingen), anti-Rab7 (1:100, Santa Cruz Biotech) or anti-β1 integrin (1:100, R&D Systems) antibodies diluted in permeabilization buffer, followed by either donkey anti-mouse Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch), donkey anti-rabbit Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch) or donkey anti-goat Cy3-labeled secondary antibody (1:100, Jackson ImmunoResearch) diluted in permeabilization buffer. Nuclei were stained using DAPI (300 nM, Life Technologies).
Anti lamp1
Manufactured by BD
Sourced in United States
Anti-LAMP1 is a laboratory equipment product that functions as an antibody targeting the LAMP1 protein. LAMP1 is a lysosomal membrane protein commonly used as a marker for lysosomes. The Anti-LAMP1 product can be utilized in various cell and molecular biology applications to detect and study the lysosomal compartment.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 goat anti mouse secondary, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Anti lamp2, by BD (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Ultraview vox spinning disk confocal system, by PerkinElmer (2 mentions)
Csu x1 spinning disk head, by Hamamatsu Photonics (2 mentions)
C9100 13, by Nikon (2 mentions)
Alexa fluor 488 goat anti rabbit secondary, by Thermo Fisher Scientific (2 mentions)
Volocity software, by PerkinElmer (2 mentions)
P35g 1.5 20 c, by MatTek (2 mentions)
35 mm glass bottomed dishes, by MatTek (2 mentions)
Anti eea1, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti calnexin, by Enzo Life Sciences (1 mentions)
Anti giantin, by Abcam (1 mentions)
Anti tgn46, by Merck Group (1 mentions)
Anti rab11, by Cell Signaling Technology (1 mentions)
Anti rab7, by Santa Cruz Biotechnology (1 mentions)
Anti β1 integrin, by R&D Systems (1 mentions)
14 protocols using anti lamp1
1
Immunofluorescence Assay for Organelle Markers
2
Western Blot Analysis of Membrane Proteins
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Pulldowns samples were eluted, boiled in Laemmli buffer and resolved by SDS-PAGE in 7%, 12% or 15% acrylamide-bisacrylamide gels or in Mini-PROTEAN TGX Gels (Bio-Rad) then, the gels were then transferred to a nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo Transfect Pack (Bio-Rad) and the Trans-Blot Turbo system (Bio-Rad) and detected with the corresponding antibodies anti-GFP (Santa Cruz Biotechnology), anti-NPC1 (Abcam), anti-NPC2 (Abcam), anti-Lamp1 (BD biosciences), anti-Lamp2 (BD bioscience), anti-PIKfyve (Abnova), anti-Flag (Sigma), anti-HA (Invitrogen), and anti-Tubulin (Sigma), GAPDH (Abcam) or HSP90 (Palex) as loading controls in Western blot (WB) analysis. Anti-mouse IgG (GE Healthcare, Chicago, IL, USA) or anti-rabbit IgG (Bio-Rad) conjugated to horseradish peroxidase was used at a dilution of 1:5000 was used as secondary antibody. Finally, bands obtained after development with ECL reagent were detected on a Molecular Imager Chemidoc XRSplus imaging system. The bands were quantified by densitometry and the data normalized to control values using Image lab software (Bio-Rad).
3
Immunohistochemical Analysis of Alzheimer's Markers
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Fixed tissue was cryoprotected and serially sectioned at a thickness of 10–20 μm. The tissue sections were immunolabeled using 6E10 antibody (Covance), anti-calbindin D28k (Sigma-Aldrich), anti-GluR1 (from Millipore or developed as described [88 (link)]), anti-LAMP1 (BD Pharmingen; San Jose, California, USA), anti-cathepsin B (Millipore), anti-synaptophysin (Millipore), and antibodies that selectively label Aβ38 and Aβ42 (Covance). Immunofluorescence analyses used appropriate Invitrogen secondary antibodies (Thermo Fisher Scientific; Waltham, Massachusetts, USA), and images were captured with a Zeiss fluorescence microscope system (Carl Zeiss, Inc.; Thornwood, New York, USA) and with a Nikon C2 point-scanning confocal microscope with NIS-Elements AR software (Nikon Instruments; Melville, New York, USA). Other images were produced via avidin–biotin–peroxidase protocols (Vector Laboratories; Burlingame, California, USA) that used 3,3′-diaminobenzidine as the chromogen. In each case, treatment groups were immunostained together and analyzed under the same instrument settings. Equally spaced coronal sections along the rostral–caudal axis of the hippocampus were used to determine the average immunoreactivity intensity and area of staining across four different view-fields.
4
Correlative Light-Electron Microscopy of Cellular Organelles
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Using a modified method (77 (link), 78 (link)), we washed cells 3 times for 5 minutes with PBS with 0.02 M glycine before incubating for 1 hour with anti-LAMP1 (Pharmingen, 1:50). We then washed cells 3 times for 10 minutes with PBS with 0.02 M glycine. The grids were incubated for 20 minutes with a goat anti–mouse Alexa Fluor 488 secondary antibody (Life technologies, Thermo Fisher Scientific, 1:500) and for 10 minutes with 1 mg/mL Nile red (MilliporeSigma, 1:25) and Hoechst 33342 (Thermo Fisher Scientific, 1:50). Finally, the grids were washed 5 times with PBS. The grids were mounted in between a glass slide and a coverslip in a droplet of VECTASHIELD and then imaged (Leica DM6, 100× oil objective). After wide-field imaging, the coverslip was removed, and the VECTASHIELD was washed from the grid with milliQ water at 37°C. Thereafter, the grids were contrasted using uranyl acetate in 2% methylcellulose for 5 minutes, blotted, and imaged (Fei Tecnai 120kV) with correlation (ICY Software) and CLEM (Huygens) deconvolution software.
5
Immunofluorescence Imaging of Cell Adhesion Proteins
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The following antibodies were used for immunofluorescence (IF): anti-E-cadherin (1:100; Cell Signaling), anti-β-catenin (1:100; Sigma-Aldrich), anti-Lamp1 (1:100; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; Life Technologies). IF was performed as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek) and were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was performed using Volocity software (PerkinElmer).
6
Immunofluorescence Microscopy of Cellular Targets
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Immunofluorescence was performed on cells cultured on glass‐bottom dishes (P35G‐1.5‐20‐C, MatTek, Ashland, MA, USA), as described previously.7 Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20℃, followed by three 5‐min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 h, followed by incubation with primary antibodies at 4℃ overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (D1306, Life Technologies, Carlsbad, CA, USA). Confocal microscopy was performed with the Ultraview Vox spinning‐disk confocal system (PerkinElmer) equipped with a Yokogawa CSU‐X1 spinning disk head and an electron‐multiplying charge‐coupled device camera (Hamamatsu C9100‐13) coupled to a Nikon Ti‐E microsope; image analysis was done using Volocity software (PerkinElmer). The following antibodies were used for immunofluorescence: anti‐phospho‐mTOR (Ser2448) (5536, Cell Signaling, Beverly, MA, USA), anti‐LAMP1 (555798, BD Biosciences, San Jose, CA, USA), anti‐BrdU (5292, Cell Signaling, Beverly, MA, USA), Alexa Fluor 568 goat anti‐mouse secondary (A‐11031, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 488 goat anti‐rabbit secondary (A‐11034, Life Technologies, Carlsbad, CA, USA).
7
Immunofluorescence Imaging of BMDC Markers
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BMDCs were cultured on #1.5 LabTek II eight-chambered coverslips (Nunc) for at least 30 minutes to allow firm adhesion. Cells were fixed by addition of ice-cold fixative (4% paraformaldehyde and 0.5% glutaraldehyde in PBS) and incubated for 30 min at room temperature in the dark, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min. Cells were then cultured with 10 µg/ml primary antibodies (Abs) in IF buffer (TBS plus human serum cocktail) overnight at 4°C in a humidifier chamber. Primary Abs consist of a mouse monoclonal anti-I-Ab (Biolegend), rat polyclonal anti-LAMP1 (BD Biosciences), and rabbit polyclonal anti-WASH and anti-VPS35 antibodies [7] (link), [16] (link), [42] (link) diluted in IF buffer. Following 5–6 washes in PBS, cells were incubated with secondary Abs (1∶500 dilution in imaging buffer) for 1 hr at room temperature. After 5–6 washes with PBS and addition of Hoechst 33342 nuclear stain, SlowFade Gold antifade reagent (Molecular Probes) was added to the wells. Images were obtained with an LSM-710 laser scanning confocal microscope with a 100X/1.4 Oil Plan-Aprochromat objective lens using ZEN software (Carl Zeiss). Each image represents an individual slice taken from a z-stack comprised of several slices at 0.25 µm depth.
8
mTOR Translocation Visualization in TSC2-/- p53-/- MEFs
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Immunofluorescence to detect mTOR translocation was performed as previously described [23 (link)]. TSC2−/−p53−/− MEFs (kindly provided by Dr. John Blenis, Weill Cornell Medical College) were seeded onto fibronectin-coated chamber slides. Cells were starved for serum over night and deprived of amino acids for 4 h using a media without any amino acids. After re-stimulating with amino acid for 1 h, the cells were fixed with 4% formaldehyde, and, subsequently, permeabilized using 0.05% saponin in PBS. Slides were treated with blocking solution (5% bovine serum albumin), and incubated with primary antibodies (anti-mTOR: Cell signaling, anti-LAMP1: BD pharmingen, San Jose, CA, USA) overnight at 4 °C followed by secondary antibodies conjugated with Alexa488 and Alexa568. Images were captured with Carl Zeiss LSM700 confocal laser scanning microscope and measured using ZEN microscope software.
9
EGF Trafficking and Receptor Dynamics
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The reagents used in this study are as follows: human EGF, Rhodamine-labeled EGF, dextran–Oregon-Green (Life Technologies) and MG132 (Sigma). The following antibodies were used in this study: anti-LAMP1, anti-LAMP2, anti-EEA1 (BD Biosciences), rabbit anti-LAMP1 (Novus Biologicals), anti-Rab7 (Cell Signaling Technology), anti-Vps41, anti-EGFR, anti-GAPDH (Santa Cruz Biotechnology), anti-Vps11 (Abcam), anti-Vps33a (Proteintech), anti-EGFR, anti-TfR, anti-Myc (Life Technologies), anti-β-actin, anti-tubulin, anti-Flag (Sigma), anti-HA (Covance) and anti-His (Pierce). Alexa-Fluor-conjugated secondary Goat anti-mouse-IgG and goat anti-rabbit-IgG antibodies were purchased from Life Technologies. Goat anti-mouse-IgG and anti-rabbit-IgG horseradish-peroxidase-conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories.
10
Muscle Fiber Isolation and Immunostaining
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Muscle fixation, isolation of single fibers, and immunostaining were performed as described,80 (link) with some modification. Briefly, isolated gastrocnemius muscles were fixed in 4% paraformaldehyde for 1 h at RT, washed with PBS, and incubated in cold methanol for 6 min at −20°C. The samples were then rinsed again with PBS and placed in a puddle of 0.04% saponin in PBS on a Sylgard-coated plate. Fibers were gently isolated from muscle bundles under a dissecting microscope. The isolated fibers were stained with anti-LC3 (Sigma-Aldrich, St. Louis, MO, USA) and anti-LAMP1 (BD Biosciences, Franklin Lakes, NJ, USA) antibodies using a M.O.M. kit (Vector Laboratories, Burlingame, CA, USA). The images were taken on a BZ-X710 microscope (Keyence, Itasca, IL, USA).
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