NRK52E cells grown on coverslips were fixed with 4% paraformaldehyde. Methanol was used for the permeabilization. Cells were washed three times with 0.1% Triton X-100 in phosphate-buffered saline (PBS), incubated overnight at 4 °C in PBS containing 0.1% Triton X-100 and 3% bovine serum albumin to block nonspecific interactions, and then incubated with anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), Plk2 (Santa Cruz Biotechnology, sc-374643), p21cip1 (Santa Cruz Biotechnology, sc-6246), and p21cip1 (MyBioSource, San Diego, CA, USA; MBS440016) antibodies. The cells were washed three times with PBST (0.1% Triton X-100) and then incubated with fluorescein isothiocyanate (FITC)–conjugated anti-rabbit secondary antibodies (Invitrogen), fluorescein isothiocyanate (FITC)–conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA, USA), Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories), or Cy3-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) and 4′, 6-diamidine-2-phenylindole (DAPI) (Sigma-Aldrich) for staining nuclear DNA. Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan).
Cy3 conjugated anti mouse secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated anti-mouse secondary antibody is a laboratory reagent used for detecting and visualizing mouse primary antibodies in various immunoassays and imaging techniques. The Cy3 fluorescent dye is conjugated to the anti-mouse secondary antibody, allowing for the detection and localization of mouse antigens or targets.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Rabbit polyclonal anti aif 1 antibody, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Fitc conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
4 6 diamidine 2 phenylindole dapi, by Merck Group (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
P21cip1, by Santa Cruz Biotechnology (1 mentions)
Avidin alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Pfk118 310, by Merck Group (1 mentions)
Mouse anti cd166, by Abcam (1 mentions)
Rabbit anti β catenin, by Cell Signaling Technology (1 mentions)
Rabbit anti cd44, by Abcam (1 mentions)
Rabbit anti cd133, by Cell Signaling Technology (1 mentions)
Goat anti actin, by Santa Cruz Biotechnology (1 mentions)
Icg 001, by Selleck Chemicals (1 mentions)
13 protocols using cy3 conjugated anti mouse secondary antibody
1
Nrf2, Plk2, and p21 Protein Localization
2
FISH Mapping of BAC Probes
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Chromosomal location of individual BAC-probes was confirmed by FISH on chicken metaphase chromosomes following standard procedures (52 ) (data not shown). FISH in interphase nuclei was performed as described in (30 (link)). Briefly, slides were denatured in 2×SSC-buffered 70% formamide during 20 min at 70°C, dehydrated in ice-cold ethanol series and air-dried. Probe mixes were denatured at 96°C for 10 min and pre-annealed at 37°C for 1 h. Immediately after pre-annealing probes were applied to denatured slides, sealed by rubber cement and hybridized at 37°C for 1–2 days. Slides were washed in 2×SSC (5 min) and 0.2×SSC 60°C (15 min), probe detection was carried out as described before (32 (link)). Biotin-labeled probes were detected by avidin-Alexa488 (ThermoFisherScientific) with signal-amplification by biotinylated anti-avidin antibodies (Jackson ImmunoResearch). Digoxigenin-labeled probes were detected with Cy3-conjugated mouse anti-digoxigenin antibodies (Jackson ImmunoResearch) with signal amplification by Cy3-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch). Finally, slides were dehydrated in ethanol series, air dried and mounted (65% glycerol, 2% 1,4-diazabicyclo[2.2.2]octane [DABCO], 1 μg/ml 4′,6-diamidino-2-phenylindole [DAPI]) for microscopy.
3
Characterization of Cancer Stem Cell Markers
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PFK118–310 was purchased from Sigma-Aldrich while ICG-001 was obtained from Selleck Chemicals. Alexa fluor 488 phalloidine and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. The antibodies used for Western blot analysis and immunocytochemistry were: rabbit anti-CD44 (Abcam # 51037), rabbit anti-CD133 (Cell Signaling # 3663), mouse anti-CD166 (Abcam # 175422), rabbit anti-Lgr5 (Sigma-Aldrich # HPA012530), goat anti-actin (Santa Cruz # sc-1615), rabbit anti-beta-catenin (Cell Signaling # 8480), mouse antibodies specific for the active form of beta-catenin, dephosphorylated on Ser 37 and Thr 41 (Millipore, clone 8E7, # 05–665). Anti-rabbit IgG horseradish peroxidase-linked antibodies and anti-mouse IgG horseradish peroxidase-linked antibodies were from Cell Signaling whereas Cy3-conjugated anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch Labs.
The following antibodies were used for flow cytometry analysis: phycoerythricine-conjugated mouse anti-human ABCB1 (BD Biosciences # 557003), phycoerythricine-conjugated mouse anti-human ABCG2 (BD Biosciences # 561180), mouse anti-human ABCC1 (BD Biosciences # 557594) and anti-mouse phycoerythricine-conjugated secondary antibodies (BD Biosciences # 555574).
The following antibodies were used for flow cytometry analysis: phycoerythricine-conjugated mouse anti-human ABCB1 (BD Biosciences # 557003), phycoerythricine-conjugated mouse anti-human ABCG2 (BD Biosciences # 561180), mouse anti-human ABCC1 (BD Biosciences # 557594) and anti-mouse phycoerythricine-conjugated secondary antibodies (BD Biosciences # 555574).
4
Immunofluorescence and Morphometric Analysis
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Immunofluorescence and morphometric analyses were executed as described below. SH-SY5Y cells were plated with a density of 75 × 103/well in a 24 wells plate, grown on a glass coverslip (coated with poly-l-lysine, Sigma-Aldrich); IMR-32 cells were plated with a density of 50 × 103/well and grown on a glass coverslip coated with collagen IV (BD Bioscience). Cells were fixed in ice-cold methanol (Sigma-Aldrich), then washed and incubated in Phosphate Buffered Saline (PBS, Sigma-Aldrich) containing 1% of Bovine Serum Albumin (BSA, Sigma-Aldrich) and 0.2% Triton X 100 overnight at 4 °C with a polyclonal anti-β III tubulin (Sigma-Aldrich, 1:600), monoclonal anti-Notch 1 (Sigma-Aldrich, 1:1000). After rinses, cells were incubated with Alexa Fluor® 488 (Life Technologies, 1:400) anti rabbit secondary antibody and CY™3-conjugated anti-mouse secondary antibody (Jackson Immunoresearch Laboratories INC.,1:500) in PBS for 1 h at room temperature. For morphological evaluation, slices were mounted and examined by a ZEISS LSM 510 META confocal laser-scanning microscope (Carl Zeiss, Germany). Images were processed using LSM5 image examiner software (Zeiss). The percentage of morphologically differentiated cells was determined by analyzing at least 10 fields for each treatment; cells with neurites ≥50 μm in length were considered as differentiated.
5
Visualizing RLuc Protein Localization
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Transfected HEK-293T cells were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (10 min incubation). Cells were treated for 1 h with PBS containing 1% bovine serum albumin and labeled overnight with the mouse anti-RLuc primary antibody (1/50; Millipore), and subsequently treated with a Cy3-conjugated anti-mouse secondary antibody [1/200; Jackson ImmunoResearch (red)] IgG (1 h each). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed in a Leica SP2 confocal microscope (Leica Microsystems). YFP-fused protein were detected by the yellow fluorescence. Nuclei were stained with Hoechst (SigmaAldrich, 1/100). Scale bar: 20 μm.
6
Immunohistochemical Profiling of Hippocampal Cells
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One out of every eight free-floating sections from the hippocampal formation was incubated with one of the following primary antibodies: rabbit polyclonal anti-AIF-1 antibody (1:300; Thermo Fisher), mouse monoclonal anti-NeuN antibody (1:250; Merck Millipore), rabbit polyclonal anti-GFAP antibody (1:400; Abcam), mouse monoclonal anti-GAD67 antibody (1:500; Merck Millipore), or rabbit monoclonal Ki-67 antibody (1:100; Thermo Scientific). Sections were then incubated for 2 h at room temperature with a Cy3-conjugated anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for AIF-1, GFAP, and Ki-67 immunohistochemistry, and with a Cy3-conjugated anti-mouse secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for GAD67 and NeuN immunohistochemistry. Nuclei were counterstained with Hoechst-33342 (Sigma-Aldrich).
7
Immunofluorescence Analysis of Cultured HPMCs
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Cultured HPMCs were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. After blocking in 2% BSA (Invitrogen) for 30 min, the cells were incubated with primary antibodies at 4 °C overnight and then incubated with FITC-conjugated anti-rabbit secondary antibody (1:1000, Invitrogen) and Cy3-conjugated anti-mouse secondary antibody (1:1000, Jackson ImmunoResearch). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1:5000, Sigma) for 5 min. Positive staining was observed under a fluorescence microscope (Olympus, Japan).
8
Fibronectin-Mediated C2C12 Cell Adhesion and Migration
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C2C12 cells were seeded at 5.000 cells cm−2 onto fibronectin-coated glass cover slips and in the presence of 10% FBS to allow cells to reorganize the adsorbed fibronectin. After 3 h of culture, cells were washed with DPBS (Gibco) and fixed in 4% formaldehyde solution (Sigma-Aldrich) for 30 min at 4 °C. Following the same procedure explained in the immunofluorescence assay section, samples were incubated with primary antibody against fibronectin (Sigma-Aldrich, 1:400) and Cy3-conjugated anti-mouse secondary antibody (Jackson Immunoresearch, 1.200) and BODIPY FL phallacidin (invitrogen, 1:100) for visualization of fibronectin and cell cytoskeleton. Nuclei were counterstained with Dapi. Nikon Eclipse 80i fluorescent microscope was used for cellular imaging. Three biological replicas were analyzed.
For migration experiments, C2C12 cells were starved overnight for synchronization (ultra-low serum conditions 1% FBS). 5.000 cells cm−2 were seeded onto fibronectin-coated glass cover slips and in the absence of FBS. The plate was disposed in the monitored staged of a Leica DMI6000 time-lapse microscope for recording of cell movements. Images were recorded every 10 min for 24 h. Six migrating cells from two time-lapse videos per condition were analyzed using the imageJ software.
For migration experiments, C2C12 cells were starved overnight for synchronization (ultra-low serum conditions 1% FBS). 5.000 cells cm−2 were seeded onto fibronectin-coated glass cover slips and in the absence of FBS. The plate was disposed in the monitored staged of a Leica DMI6000 time-lapse microscope for recording of cell movements. Images were recorded every 10 min for 24 h. Six migrating cells from two time-lapse videos per condition were analyzed using the imageJ software.
9
Quantifying Hippocampal Neurons and Microglia
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One out of every 8 serial brain sections from the hippocampal formation was incubated with one of the following primary antibodies: mouse monoclonal anti-NeuN antibody (1:250; Merk Millipore, Burlington, MA, USA) or rabbit polyclonal anti-AIF-1 antibody (1:300; Thermo Fisher Scientific, Waltham, MA, USA). Sections were then incubated for 2 h at room temperature with a Cy3-conjugated anti-mouse secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or with a Cy3-conjugated anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Nuclei were counterstained with Hoechst-33342 (Sigma-Aldrich, St. Louis, MO, USA). Fluorescent images were acquired using a Nikon Eclipse TE600 microscope equipped with a Nikon Digital Camera DXM1200 ATI System (Nikon Instruments Inc., Melville, NY, USA). The number of Hoechst-positive and NeuN-positive cells were manually counted using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA) and established as cells/mm3. Starting from 20X magnification images of AIF-1-stained hippocampal slices, AIF-1 positive microglial cell body size was manually drawn using the Image Pro Plus measurement function, and expressed in µm2.
10
Immunostaining of Dopaminergic Neurons in Rat Brains
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Immunohistostaining was performed as described in [39 (link)] with modifications. Briefly, adult rats were anesthetized with KAX (86.9 mg/ml Ketamine, 0.43 mg/ml Acepromazine and 5.22 mg/ml Xylazine) at a dosage of 1 μl/gram body weight through intraperitoneal injection and then perfused with 10% neutral buffered formalin (NBF) (Sigma-Aldrich). Brains were dissected and post-fixed in 10% NBF overnight, thoroughly washed and soaked in 30% sucrose in 1XPBS until they sank. They were then embedded into Optimal Cutting Temperature compound (Tissue-Tek) and cut into 8μm thick frozen sections. Before incubated with primary antibodies, brain sections were permeabilized with 0.1% Triton X-100 in 1XPBS at room temperature for 20 minutes and blocked in Blocking buffer (3% BSA, 0.1% tween in 1XPBS) for 1 hour. Antibody incubation was performed at 4°C overnight. Mouse anti-tyrosine hydroxylase (clone LNC1, Millipore) monoclonal antibody was used at 1:400. Following washing steps with 1XPBS, Alexa Fluor 488 or Cy3-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) was used to detect the signal of the primary antibody. For Rosa Tom reporter, the live tdTomato signal was acquired with Cy3 filter. At least three animals from each genotype were analyzed.
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