Rabbit polyclonal antibody against LC3 was purchased from MBL (PM036; Nagoya, Japan), and rat monoclonal antibody against LAMP1 (ab25245) was obtained from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against LAMP1 (sc-20011) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Biotinylated anti-rabbit immunoglobulin antibody (E0432; DAKO, Glostrup, Denmark), FITC-conjugated streptavidin (F0422; DAKO), Alexa647 conjugated anti-rat immunoglobulin antibody (A-21247; Molecular Probes, Waltham, MA, USA), and Alexa647 conjugated anti-mouse immunoglobulin antibody (115-606-146; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used for detection.
Sc 20011
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-20011 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for general laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab56416, by Abcam (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Ab50533, by Abcam (2 mentions)
Lamp1, by Santa Cruz Biotechnology (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab25245, by Abcam (1 mentions)
E0432, by Agilent Technologies (1 mentions)
Fitc conjugated streptavidin, by Agilent Technologies (1 mentions)
Gtx70228, by GeneTex (1 mentions)
Sc 135992, by Santa Cruz Biotechnology (1 mentions)
Nbp1 49955, by Novus Biologicals (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Sc 18822, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sc 8017, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)
Lysotrakerred, by Thermo Fisher Scientific (1 mentions)
21 protocols using sc 20011
1
Immunofluorescence Detection of Autophagy Markers
2
Immunofluorescence Staining of DNA Repair and Autophagy Markers
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Cells were grown on coverslips, fixed in −20 °C 100% methanol for 10 min on ice, and permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were incubated with blocking buffer (1% BSA, 0.1% Tween 20 in PBS) for 1 h at RT. Coverslips were incubated with primary antibodies for 1 h at RT, washed three times with 1% PBS and once with blocking buffer, and incubated with the appropriate fluorescent secondary antibody for 1 h at RT. The coverslips were then washed three times with 1% PBS and mounted on microscopy slides using mounting reagent with DAPI (Ibidi GmbH, Gräfelfing, Germany). Staining was analyzed using a Leica DMi8 inverted microscope with a 63x oil-immersion objective using the same laser parameters. All microscope images were analyzed with ImageJ software. Primary antibodies used: anti-RAD50 mouse monoclonal antibody (GTX70228, GeneTex), anti-NBS1 rabbit monoclonal antibody (#14956, Cell Signaling Technology), anti-MRE11 mouse monoclonal antibody (sc-135992, Santa Cruz Biotechnology), anti-p62 rabbit polyclonal antibody (NBP1-49955, Novus Biologicals), anti-LC3 rabbit polyclonal antibody (NP100-2220, Novus Biologicals) and anti-LAMP1 mouse monoclonal antibody (sc-20011, Santa Cruz Biotechnology).
3
Western Blot Analysis of Lysosomal Proteins
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Total proteins were separated electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Specific primary antibodies against LAMP1 (sc-20,011, Santa Cruz Biotechnology, USA), LAMP2 (sc-18,822, Santa Cruz Biotechnology, USA), LC3B (#3868, Cell Signaling Technology, USA), p62 (ab56416, Abcam, USA); together with appropriate HRP-linked secondary antibody were used. β-actin was used as a loading control.
4
Immunofluorescence Quantification of CSTD, LAMP1, and Ubiquitin
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Cells were seeded in 96-well black plates. Treatments were performed as described previously. The fixation step protocol was followed [25 (link)]. After permeabilization, the cells were incubated with primary antibodies against CSTD (D-7) (sc-377299, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200), LAMP1 (H4A3) (sc-20011, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200) and ubiquitin (P4D1) (sc-8017, Santa-Cruz Biotechnology, Paso Robles, CA, USA, 1:200) for 1–2 h at room temperature and then incubated with Alexa Fluor® 568 (A11004)- or 488 (A11008)-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Image quantification was conducted with Image J software (National Institute of Health, Bethesda, MD, USA).
5
Quantifying Lysosomal Aggregation in Cells
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SORLA-silenced and control-silenced cells were fixed and stained for LAMP1 (Santa-Cruz, SC-20011) and DAPI as described above. Cells were imaged with identical microscope settings with a Carl Zeiss LSM780 laser scanning confocal microscope. Image processing and quantifications were performed with the ImageJ software. The level of LAMP1-positive late-endosome/lysosome aggregation was quantified from the middle plane. To distinguish between individual late-endosome/lysosome aggregates, watershed segmentation was used before quantification. The areas of late-endosome/lysosome aggregates were quantified from a single cell. A LAMP1-positive area larger than 5 µm2 was considered as aggregate. Total area of LAMP1-positive late endosomes/lysosomes was calculated based on LAMP1 staining as well as the area of aggregates larger than 5 µm2. The percentage of lysosomal aggregation was determined by the ratio of area of aggregates larger than 5 µm2 to total LAMP1-positive late-endosome/lysosome area.
6
Visualizing Autophagosomes and Lysosomes
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Cells grown on coverslips were fixed in 3.7% paraformaldehyde with phosphate-buffered saline (PBS) for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, blocked with 10% FBS in PBS for 2 h, and incubated with primary antibodies overnight. Antibodies against human LC3 (#2775) or Lamp1 (SC-20011; Santa Cruz Biotechnology, USA) detected autophagosomes or lysosomes, respectively. In addition, cells were washed and incubated with Alexa Fluor 488-conjugated anti-rabbit, 633-conjugated anti-mouse, 488-conjugated anti-mouse, 405-conjugated anti-mouse, 546-conjugated anti-rabbit, or 546-conjugated anti-mouse secondary antibodies (all from Thermo Fisher Scientific) for 2 h and visualized under a confocal microscope (LSM 510; Carl Zeiss, USA). Alternatively, lysosomes were directly labeled by using LysoTrakerRed (L7528; Thermo Fisher Scientific). Lipofuscins were visualized from cells grown on coverslips through confocal microscopy and autofluorescence at a 505-555 nm wavelength and photographed using a FITC filter after a 493 nm excitation. ImageJ analysis software (NIH, USA) counted puncta in the fluorescence images over 0.5 μm2.
7
Multiparameter Immunofluorescence Staining
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The cells were fixed in 4% paraformaldehyde for 5 min and blocked with 5% donkey serum albumin at room temperature for 30 min. They were then incubated with the primary antibodies at 4 °C overnight. The primary antibodies used for staining were as follows: mouse anti-hBcl2 (1:100, NBP2-15200, Novus, USA), rabbit anti-cleaved caspase3 (1:100, ab32042, Abcam, USA), and mouse anti-Lamp1 (1:100, sc20011, Santa Cruz, USA). The samples were incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies used were as follows: donkey anti-mouse Alexa Fluor 488, donkey anti-mouse Alexa Fluor 647, donkey anti-mouse Alexa Fluor 594, and donkey anti-rabbit Alexa Fluor 594 (1:500, Termo Fisher Scientific, USA). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, C1005, Beyotime Biotechnology, China) was used for nuclear staining.
8
Visualizing Lysosomal Dynamics in Cells
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The cells were fixed and stained with primary antibodies against LAMP-1 (sc-20011, Santa Cruz Biotechnology) and cathepsin B, followed by a secondary antibody conjugated with Alexa Fluor 488 or 546. Nuclei were counterstained with (DAPI; 10236276001; Roche, Mannheim, Germany). Live cells were stained with 5 μg/mL acridine orange at 37 ℃ for 30 min. Fluorescent images were obtained using a laser-scanning confocal microscope (LSM800; Carl Zeiss, Oberkochen, Germany).
9
Quantifying CD71 and LAMP1 in Aspergillus-Infected Cells
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RCB2280 cells were cultured in 22 × 22mm coverslips and mixed with conidia (CI1698) at a multiplicity of ten and incubated for 6 h [25 (link)]. Following infection, cells were washed with 1X PBS, fixed with optimized fixation and permeabilization methods for CD71 (4% PFA and 0.1% triton X-100) and LAMP1 (MeOH-Acetone). After blocking, cells were incubated with mouse monoclonal antibodies of CD71 (transferrin receptor) (sc-65882, Santa Cruz Biotechnology, Texas, USA) or LAMP1 (sc-20011, Santa Cruz Biotechnology, Texas, USA) (1:50) for 1 h at room temperature. Finally, the cells were stained with goat anti-mouse IgG conjugated with dylight 550 (1:50) for 1 h at room temperature. Coverslips were mounted on glass slides using vectashield mounting medium containing DAPI and sealed with nail polish. The images were acquired using Leica TCS SP8 confocal laser scanning microscopy and analyzed using LAS AF Lite software.
10
Intracellular Trafficking of BACE1 in Neurons
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Cells were fixed using 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% FBS for 1 h. Cells were incubated with primary antibodies overnight at 4 °C and labeled with secondary antibodies at 37 °C for 1 h. Images were acquired using confocal fluorescence microscopy (LSM800, ZEISS). For making frozen sections, mice were anesthetized with sodium pentobarbital and perfused with PBS, followed by 4% paraformaldehyde. Following perfusion, the brain was obtained and post-fixed overnight in fixative solution, then cryoprotected overnight in 30% sucrose in PBS. Frozen brain tissues were embedded in TissueTek OCT compound, then cut into 10 μm sections. The sections were observed under confocal fluorescence microscopy (LSM800, ZEISS). BACE1 (1: 200, 3C1C3; 1:200, ab183612) antibody was purchased from Invitrogen or Abcam. EEA1 (1:200, ab2900) antibody and Rab7 (1:200, ab50533) antibody were purchased from Abcam. LAMP1 (1:100, sc-20011) antibody was purchased from Santa Cruz Biotechnology. Alexa Fluor 568 (1:500) and Alexa Fluor 647 (1:500) secondary antibody was purchased from Abcam.
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