293T cells were grown on Lab-Tek 2 well chamber slide (Nunc, Thermo) at 2 × 104 cells per well and transfected the next days using TransIT-LT1 transfection reagent. Two days after transfection, the cells were fixed in 3.6% formaldehyde in 1× phosphate-buffered saline (PBS), permeabilized in 0.1% NP-40 in 1× PBS at room temperature, and incubated with anti-HA antibody (HA-7) at a 1:300 dilution in 1× PBS containing 3% bovine serum albumin (BSA) at 37°C for 30 min. Cells were then stained with Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a 1:300 dilution in 1× PBS containing 3% BSA at 37°C for 30 min. Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole). Following washing three times in 1× PBS, the coverslides were mounted on slides using SlowFade Gold antifade reagent (Life Technology). Samples were analyzed under a confocal laser-scanning microscope (FV1200; Olympus, Tokyo, Japan).
Cy3 conjugated anti mouse antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated anti-mouse antibody is a laboratory reagent used to detect and quantify mouse proteins in various applications. It consists of a mouse-specific antibody molecule conjugated to the Cy3 fluorescent dye, which enables visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Fluorescein 488 conjugated anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Fitc conjugated anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Cy3 conjugated anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Apotome microscope, by Zeiss (2 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
Mouse anti β galactosidase, by Promega (1 mentions)
Sc 28759, by Santa Cruz Biotechnology (1 mentions)
Ab15360, by Abcam (1 mentions)
Mnk4428, by Fujifilm (1 mentions)
Rabbit anti iba1, by Proteintech (1 mentions)
Rabbit anti cd206, by Abcam (1 mentions)
Rabbit anti zo 1, by Abcam (1 mentions)
Mouse anti iba1, by Abcam (1 mentions)
Rabbit anti inos, by Abcam (1 mentions)
Tetramethylrhodamine isothiocyanate tritc conjugated phalloidin, by Merck Group (1 mentions)
Mouse monoclonal anti alpha tubulin, by Merck Group (1 mentions)
16 protocols using cy3 conjugated anti mouse antibody
1
Immunostaining of HA-tagged Proteins in 293T Cells
2
Immunohistochemistry Protocol for Neuronal Markers
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Primary antibodies used for immunohistochemistry were: Mouse anti-β-galactosidase (1:5000; ref 18637314, Promega), rabbit anti-TRPV1 (1:1000, ref sc-28759, Santa Cruz), rabbit anti-CGRP (1:1000, ref AB15360, Abcam), rabbit anti-substance P (1:500, ref 84500505, Biogenesis Ltd), rabbit anti-TrkA (1:500, ref SAB1305371, Sigma), goat anti-c-ret (1:500; ref AF482, R&D System), rabbit anti-phospho GluN1 (Ser896, 1:1000; Upstate), rabbit anti-phospho-Erk (1:1000, ref 9101, Cell Signalling), rabbit anti-HA (1:500, ref 715500, Life Technologies), mouse anti-Iba1 (1:2000, ref MNK4428, Wako), Griffonia simplicifolia isolectin (IB4) FITC conjugated (1:1000, Sigma). Secondary antibodies were Alexa Fluor 488 anti-rabbit or anti-goat antibody (1:2000) from Life Technologies, and Cy3-conjugated anti-mouse antibody (1:2000) was from Jackson ImmunoResearch Laboratories.
3
Immunofluorescence Analysis of Brain Tissue
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The paraffin section (5 μm-thick) of the brain was prepared, after being fixed, washed, permeabilized, and blocked, cells were incubated with rabbit anti-ZO-1 (1:200, Abcam), or rabbit anti-Iba-1 (1:100, Proteintech). After that, sections were further incubated with the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch). Slides were imaged using a fluorescence microscope. To explore the polarization reprogramming effects of DEX, we performed immunofluorescence double labeling. Sections were incubated in mouse anti-Iba-1 (1:100, Abcam), with rabbit anti-iNOS (1:100, Abcam) or rabbit anti-CD206 (1:100, Abcam) for 1 h at 37°C. Antibodies were then used: the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch, United States), or Cy3-conjugated anti-mouse antibody (1:1000, Jackson Immunoresearch).
4
Visualizing Cytoskeleton Dynamics In Cells
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Cells were seeded at a density of 4×104 cells per milliliter and grown on 4-well Lab-Tek chamber slides (Nalgene Nunc Penfield, NY, USA). Cells were fixed with absolute ethanol for 10 min and blocked using 5% bovine serum albumin in Dulbecco’s phosphate-buffered saline (DPBS, Biowest). The chamber slides were incubated overnight at 4°C with mouse monoclonal anti-alpha tubulin (1∶8000; Sigma-Aldrich, St. Louis, MO, USA). The chamber slides were then washed with DPBS (Biowest) and incubated with Cy3-conjugated anti-mouse antibody (1∶2000; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. Staining for F-actin was carried out using tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1 µg/mL; Sigma-Aldrich). Counterstaining of cell nuclei was carried out using 4′,6-diamidino-2′-phenylindole (DAPI, Roche, Germany; dilution 1∶1000) for 10 min at room temperature. Slides were viewed using a confocal microscope (LSM 700; Carl Zeiss Co. Ltd., Oberkochen, Germany).
5
Antibody Profiling for Translational Regulation
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The following antibodies were used throughout the whole study: mouse monoclonal to eIF3η C-5 (Santa Cruz Biotechnology, sc-137114, 1:500), rabbit polyclonal to DDX6 (Bethyl Laboratories, A300-461A, 1:500), rabbit polyclonal to eIF4E1 (Sigma-Aldrich, E5906, 1:200), rabbit polyclonal to eIF4E2 (Genetex, GTX82524, 1:200), rabbit polyclonal to 4E-T (kind gift from Prof. Sonenberg, 1:200), Cy™3-conjugated anti-mouse antibody and Cy™5-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories, 715-165-151, 711-175-152, 1:500), mouse monoclonal anti-β-actin antibody (A2228, Sigma-Aldrich, 1:1000), mouse monoclonal anti-GFP antibody (sc-9996, Santa Cruz, 1:1000), mouse monoclonal anti-eIF4G1 antibody (sc-373892, Santa Cruz, 1:500) and goat anti-mouse IgG-HRP (sc-2005, Santa Cruz, 1:5000).
6
Tadpole Retinal Membrane Protein Localization
7
Immunofluorescent Detection of p53
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Cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% triton, and blocked with 2% BSA. p53 was detected using a mouse monoclonal antibody (SC-126, Santa Cruz Biotechnology) followed by a Cy3 conjugated anti-mouse antibody (Jackson Immunoresearch Laboratories). Cells were stained with Dapi and viewed on an Axioimager fluorescent microscope (Zeiss).
8
Quantifying Autophagy Markers LC3B and p62
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Cells were washed in phosphate buffered saline (PBS), prefixed in 4% paraformaldehyde for 15s at room temperature (RT), fixed in ice-cold 100% methanol for 10min at 20°C and washed PBS at RT. Primary antibodies, namely anti-LC3B antibody (Cell Signaling, Lausen, Switzerland, rabbit monoclonal, clone D11, #3686) and anti-p62 antibody (Sigma, Leiden, Netherlands, mouse monoclonal, clone 2C11, #WH0008878M1) were diluted 1:100 and 1:200 in PBS/1% bovine serum albumin (BSA)/0.1% Tween, respectively. Slides were incubated with primary antibodies for 1h at RT. Slides was washed twice with PBS/0.1% Tween and once with PBS only. Secondary antibodies, namely FITC conjugated anti-rabbit antibody (Jackson Immunoresearch, Suffolk United Kingdom, #111-096-045) and Cy3 conjugated anti-mouse antibody (Jackson Immunoresearch, Suffolk, United Kingdom, #115-166-003), were diluted 1:130 in PBS/1%BSA/0.1%Tween. Slides were incubated with secondary antibodies for 1h at RT. Images were taken on Olympus FluoView microscope at 63x objective magnification and adjusted for brightness using ImageJ Software (NIH, Bethesda, MD, United States of America, 1.64r).
9
Immunohistochemical Analysis of Cerebral Microvascular Endothelial Cells
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For the immunohistochemical analysis, sections were incubated with mouse anti-VEGF (1 : 200, Abcam) or mouse anti-HIF-1α (1 : 200, Novus), and then with biotinylated anti-mouse IgG (1 : 800, Santa Cruz) for 1 h at 37°C. The samples were then incubated with an avidin-biotin-peroxidase complex (1 : 100, Vector Laboratories) for 1 h at 37°C. Immunoreactivity was visualized with DAB.
To detect proliferating cerebral microvascular endothelial cells, the sections were incubated with mouse anti-PCNA (1 : 1400, Cell Signaling Technology) and rabbit anti-vWF (1 : 400, Dakon) for 1 h at 37°C. The following secondary antibodies were then used: fluorescein 488-conjugated anti-rabbit antibody (1 : 1000, Jackson ImmunoResearch) and Cy3-conjugated anti-mouse antibody (1 : 1000, Jackson ImmunoResearch). After washing with PBS, the sections were stained with DAPI for 2 min to reveal the location of the nucleus. PCNA+/vWF+ nuclei close to the hematoma were counted. The data were presented as the number of nuclei per mm2 (N/mm2). To determine whether VEGF and HIF-1α were expressed in the endothelial cells , tissue sections were incubated with mouse anti-VEGF (1 : 200, Abcam) or mouse anti-HIF-1a (1 : 100, Novus) with rabbit anti-vWF (1 : 400, Dakon).
To detect proliferating cerebral microvascular endothelial cells, the sections were incubated with mouse anti-PCNA (1 : 1400, Cell Signaling Technology) and rabbit anti-vWF (1 : 400, Dakon) for 1 h at 37°C. The following secondary antibodies were then used: fluorescein 488-conjugated anti-rabbit antibody (1 : 1000, Jackson ImmunoResearch) and Cy3-conjugated anti-mouse antibody (1 : 1000, Jackson ImmunoResearch). After washing with PBS, the sections were stained with DAPI for 2 min to reveal the location of the nucleus. PCNA+/vWF+ nuclei close to the hematoma were counted. The data were presented as the number of nuclei per mm2 (N/mm2). To determine whether VEGF and HIF-1α were expressed in the endothelial cells , tissue sections were incubated with mouse anti-VEGF (1 : 200, Abcam) or mouse anti-HIF-1a (1 : 100, Novus) with rabbit anti-vWF (1 : 400, Dakon).
10
Neurite Length Measurement in N2a Cells
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To investigate the effect of CM collected from the cultured postnatal mouse retinal cells at each specific developmental stage, differentiation of the N2a cells was investigated by measuring the total neurite length of the N2a cells. Treated N2a cells (1×105 cells per dish) were fixed with 4% paraformaldehyde for immunocytochemistry. To visualize the neurites of the cells, primary mouse anti-neuron-specific beta-III tubulin (TuJ1; R&D Systems, Minneapolis, Minnesota, USA) and secondary Cy3-conjugated anti-mouse antibodies (Jackson ImmunoResearch Laboratories, Inc.) were used. TuJ1-positive cells were examined using a Zeiss Axioplan microscope with a Zeiss Plan-Apochromat 40× objective and a Zeiss AxioCam HRc digital camera (Carl Zeiss Meditec, Inc.). The cells were viewed on a computer monitor, and the length of the TuJ1-positive neurites was measured from the soma to the dendritic tip. All visible neurites were measured regardless of their number, except those that were shorter than the diameter of the soma. The measurement was repeated three times independently using Image J software (US National Institute of Health, Bethesda, Maryland, USA).
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