Immunofluorescence staining was performed as described previously (De Graauw et al, 2005 (link)). Primary antibody incubation was performed overnight at 4 °C, and tumours were subsequently incubated with the Alexa-488 or Cy-3-conjugated secondary antibody (Molecular Probes, Bleiswijk, The Netherlands). Microscopic analysis was performed using a Bio-Rad Radiance 2100 confocal system with a × 60 or × 40 Plan Apo (NA 1.4; Nikon) objective lens. Image acquisition was controlled using the Laser Sharp software (Bio-Rad, Hercules, CA, USA). Images were processed, and quantitative image analysis was performed using Image-Pro Plus (Version 5.1; Media Cybernetics, Rockville, MD, USA).
Cy3 conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cy3-conjugated secondary antibodies are fluorescently labeled antibodies used in immunoassays and other applications that require the detection of target proteins. They consist of a secondary antibody conjugated to the Cy3 fluorescent dye, which emits light in the orange-red spectrum when excited.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Alexa 488, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Fv1000, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
A confocal microscope, by Leica camera (2 mentions)
Fitc or, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Dingguo (2 mentions)
Axio imager m2 fluorescence microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lasersharp software, by Bio-Rad (1 mentions)
Radiance 2100, by Nikon (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Microscopy, by Olympus (1 mentions)
Fitc labeled secondary antibody, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ix71 inverted fluorescence microscope, by Olympus (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
34 protocols using cy3 conjugated secondary antibody
1
Immunofluorescence Staining for Tumor Analysis
2
Immunohistochemical Analysis of TDP-43
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Dissection, fixation, immunolabelling, and confocal imaging acquisition were executed as previously described [117 (link)]. The ENV primary antibody was used as described in Li, et al. 2013 [21 (link), 78 (link)]. For TDP-43 immunohistochemistry, the primary full length human TDP-43 antibody (Protein Tech, 10782-2-AP) was used at a 1:100 dilution, and the primary pSer409 phosphorylated human TDP-43 antibody (Sigma Aldrich, SAB4200223) was used at a 1:500 dilution separately in conjunction with a 1:200 dilution of an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, A-11070). Repo co-labeling was performed using a 1:200 dilution of primary antibody (Developmental Studies Hybridoma Bank, 8D12) and a 1:200 dilution of a Cy3-conjugated secondary antibody (Molecular Probes, A10521). DAPI co-staining was performed after a brief wash in 1x PBS immediately subsequent to secondary antibody staining using DAPI Dilactate (Thermo Fisher Scientific, D3571) as per manufacturer specifications. All brains co-stained with DAPI were imaged on a Zeiss LSM 780 confocal microscope using a UV laser and the Zeiss ZEN microscope software package.
3
Microglia TLR Expression Profiling
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BV-2 microglia cells seeded at 2.5 × 104 cells/mL on sterile cover slips were cultured in 24-well plates. BV-2 cells were pretreated with 0.1 mM MPP+ for 12 h. Then cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 for 30 min. After blocking with 5% nonfat milk in PBS buffer, cells were incubated with rabbit anti-TLR2, TLR4, and TLR9 antibodies for 1 h at room temperature. After brief wash, cells were incubated with Cy3-conjugated secondary antibody (1 : 500, Molecular Probes). Finally, the cells were washed again, mounted with VECTASHIELD hard mount with DAPI, and visualized using fluorescence microscope.
4
Retinal Histology and Immunofluorescence Protocol
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For retinal histological study, eyeballs were immersed in Per-fix (4% paraformaldehyde, 20% isopropanol, 2% trichloroacetic acid, 2% zinc chloride) overnight and embedded in paraffin. Sagittal sections of 5 μm thickness were stained with hematoxylin and eosin (HE) and examined under an Olympus microscopy (Tokyo, Japan). Immunofluorescence study on retinal cryosections was prepared as previously described [11 (link)]. Briefly, the sections were immunostained with anti-CD11b antibody (1:100 dilution) overnight at 4°C. Negative controls without primary antibody incubation were included. After multiple washes, the sections were incubated with Cy3-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). DAPI was used to label cell nuclei.
5
Characterization of Induced Pluripotent Stem Cells
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The iPS cells were fixed by 4% paraformaldehyde, and permeabilized by 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, USA). 12 (link) To characterized the iPS cells, the specimens (n = 6) were incubated overnight at 4°C with mouse anti-OCT4 (1:100), mouse anti-SSEA-4 (1:100) or mouse anti-SSEA-1 (1:100), and were then incubated with a Cy3-conjugated secondary antibody (1:100, Molecular Probes, Breda, Netherlands) for 1 h, and subsequently with 4',6-diamidino-2-phenyl indole (DAPI) for 3 min at room temperature. To evaluate the ameloblastic differentiation of iPS cells (n = 6), goat anti-ameloblastin (1:100), goat antienamelin (1:100), rabbit anti-CK14 (1:100) primary antibodies, and FITC-labeled secondary antibody (1:200; Chemicon, Billerica, USA) were used. All primary antibodies were purchased from the Santa Cruz Biotechnology (Dallas, USA). Samples (n = 6) were examined under a confocal microscope (FV1000, Olympus, Japan). Five fields of view at 400× magnification were selected at random, and the percentage of the number of immune-positive cells to total cells in each field was counted and averaged for statistical analysis.
6
Immunofluorescence Analysis of Cell-Cell Adhesion
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Cells were plated into 12-well plates at 2.0 × 104 cells per well. After 48 h, the cells were fixed in 4% paraformaldehyde for 30 min. After washing with PBS for 3 times, the cells were blocked in blocking solution (3% BSA, 0.3% Triton X-100) at room temperature for 60 min. The cells were subsequently incubated with the primary antibodies (rabbit anti-E-cadherin at 1:50; rabbit anti-β-catenin at 1:50; CST) at 4°C overnight. The primary antibodies were then removed, and secondary antibody was added (Cy3-conjugated secondary antibody at 1:500; Life Technologies), and incubated for 1 h. Finally, the nuclei were stained with 0.5 μg/mL DAPI (Sigma), and the fixed cells were observed and photographed using an Olympus IX71 inverted fluorescence microscope (Olympus Optical).
7
Immunofluorescent Labeling of NADPH Oxidase Components
8
Ki67 Immunofluorescence Assay for Cell Proliferation
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Cell proliferation was determined by ki67 immunofluorescence staining. Briefly, HRVECs were seeded in a 24‐well plate and processed with the required treatments. They were fixed in 4% formaldehyde (Biosharp) for 15 min and then blocked with 5% bovine serum albumin (BSA) (143,066, Biofroxx) for 1 h. They were incubated with ki67 antibody (Abcam) overnight at 4°C and then incubated with Cy3‐conjugated secondary antibody (Life Technologies) for 3 h at room temperature. Cell nuclei were labelled with 4′,6‐diamidino‐2‐phenylindole (DAPI, C1002, Beyotime). The images of stained cells were observed under a fluorescence microscope.
9
Immunofluorescence analysis of 3-nitrotyrosine and NF-kB in HAECs and mouse aorta
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Immunofluorescence analysis was performed on HAECs grown on gelatin-coated glass coverslips as well as 5 μm sections of paraffin-embedded mouse aortas. Samples were antigen-retrieved, fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 and washed using PBS. Sections were blocked in PBS with 2% bovine serum albumin (BSA) for 1 h at room temperature. HAECs on coverslips were then incubated with anti-3-nitrotyrosine (ab61392, Abcam, 1:100) for 1 h at room temperature, or anti-total-NFκB p65 antibody (#8242, Cell Signaling, 1:100) overnight at 4 °C followed by Cy3-conjugated secondary antibody (Life Technologies Inc., 1:1000). Coverslips were then stained for nuclei with Hoechst dye, and cover-slipped using gelvatol mounting media (polyvinylalcohol, glycerol, H2O, sodium azide and Tris pH 8.5). Nonspecific rabbit or goat IgG (5 μg/ml) was used in lieu of primary antibody as a negative control. Confocal images were captured on a Nikon A1 spectral confocal microscope (Nikon Instruments Inc. Melville, NY). For each experiment, 3-4 images per treatment group were captured. Three independent experiments were performed.
10
In vivo T cell tracking in tumor
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T cells were labeled with CFSE (Abcam, Cambridge, UK). T cells (1×107) modified with 100 µg iRGD-anti-CD3 or control reagents, or codelivered with 3 µg free iRGD were injected into subcutaneous tumor-bearing mice via the tail vein. Tumors were harvested at 24 hours after injection and processed for immunostaining. Frozen sections were stained with an anti-CD31 antibody (550274, BD Pharmingen) followed by a Cy3-conjugated secondary antibody (A10522, Life Technologies). After washing with PBS, the sections were mounted with DAPI (Beyotime, Shanghai, China) and imaged at ×200 magnification.
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