TRC105 was provided by TRACON Pharmaceuticals, Inc. Rat anti-mouse CD31 primary antibody was purchased from BD Biosciences. AlexaFluor488- and Cy3-labeled secondary antibodies were purchased from Jackson Immunoresearch Laboratories, Inc. S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. Fluorescein isothiocyanate (FITC) and chelex 100 resin (50–100 mesh) were acquired from Sigma-Aldrich. PD-10 desalting columns were purchased from GE Healthcare. All other reaction buffers and chemicals were from Thermo Fisher Scientific.
Cy3 labeled secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-labeled secondary antibody is a fluorescent-conjugated antibody used for detection and visualization in various immunological and biochemical assays. It binds to the primary antibody and emits a red-orange fluorescent signal upon excitation, allowing for the identification and localization of specific targets.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by GE Healthcare (4 mentions)
Chelex 100 resin, by Merck Group (3 mentions)
P scn bn nota, by Macrocyclics (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Scm peg5k mal, by Creative PEGWorks (2 mentions)
Sunitinib malate, by Bio-Techne (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Alexa fluor 488 and, by Jackson ImmunoResearch (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Rat anti mouse cd31 primary antibody, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
S 2 4 isothiocyanatobenzyl 1 4 7 triazacyclononane 1 4 7 triacetic acid p scn bn nota, by Macrocyclics (1 mentions)
Pepsin, by Thermo Fisher Scientific (1 mentions)
Fitc lectin, by Vector Laboratories (1 mentions)
Fshr mab, by R&D Systems (1 mentions)
Ki 67, by Abcam (1 mentions)
Histostain sp kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Nanog, by Santa Cruz Biotechnology (1 mentions)
Ssea 1, by Santa Cruz Biotechnology (1 mentions)
23 protocols using cy3 labeled secondary antibody
1
Radiolabeling of TRC105 Antibody
2
Histological Analysis of Mouse Lung Tissue
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Mice were sacrificed, the lungs were removed, fixed in 4 % PFA for 38 h, serially dehydrated, and embedded in paraffin for sectioning at 6 μm. For stainings, sections were dewaxed and rehydrated. H&E staining was performed by incubating lung sections in hematoxylin for 2 min and in eosin for 1 min. For fluorescence staining, lung sections were incubated in pepsin (Invitrogen) for 20 min at 37 °C, washed, and treated for 10 min in PBS supplemented with 5 % FCS and 0.5 % Tween 20. Lung sections were exposed overnight at 4 °C to anti-ZO1 IgG, anti-ZO2 IgG, anti-E-cadherin IgG, anti-occludin IgG, or anti-Ly-6G and Ly-6C (Gr-1, rat IgG; BD) antibodies (all antibodies as above). The sections were washed in PBS with 0.05 % Tween 20 and then incubated for an additional 45 min with Cy3-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). For co-stainings of TJs and endothelial cells, samples were incubated together with antibodies against ZO1, ZO2, occludin, and E-cadherin additionally with FITC-Lectin (Fluorescein Griffona Simplicifolia Lectin I-Isolectin B4, Vector, FL-1201, 1:50) and then proceeded as above. The sections were washed once in PBS with 0.05 % Tween 20 and once in PBS and were mounted in Mowiol. Lung samples were analyzed by light transmission or confocal microscopy.
3
Radiolabeling of FSHR-Monoclonal Antibody
4
Immunohistochemical Analysis of Cell Proliferation
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All of the fixed samples were dehydrated in a graded alcohol series. Tissue samples were eventually embedded in paraffin before being sectioned. The rehydrated sections were employed for immunostaining. Primary antibodies specific to Ki67 (Epitomics, Burlingame, CA, USA), PH3 (Cell Signaling Technology, USA) and PCNA (Cell Signaling Technology, Beverly, MA, USA) were used. Sections incubated without primary antibodies were used as negative controls. High-temperature heating was used for antigen retrieval. A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) was applied to visualize the antigen. For immunofluorescence, liver sections (8 μm) were fixed in 4% paraformaldehyde solution. Primary antibodies specific to p27 (ABclonal Technology, Wuhan, China) were used, and the sections were stained with Cy3-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) before being counterstained with 4′,6-diamidino-2-phenylindole. Immunofluorescence images were captured via a Leica TCS SP5 confocal scanning laser microscope (Leica, Solms, Germany).
5
Immunophenotyping of Induced Pluripotent Stem Cells
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For immunostaining, iPSCs (passage 5) were fixed in 4% paraformaldehyde for 20 minutes at 4°C and then washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100 for 10 minutes. After washing with PBS, the cells were incubated in 4% bovine serum albumin for 1 hour to block nonspecific binding. The cells were incubated with primary antibodies against SSEA1, Nanog, Oct4, and Sox2 (Santa Cruz Biotechnology Inc, Dallas, TX, USA) overnight at 4°C. After washing with PBS, the cells wree incubated with Cy-3-labeled secondary antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA) for 1 hour. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:2,000 dilution) (Sigma-Aldrich Corp).
6
Fluorescent Labeling of Antibodies
7
Immunofluorescent Staining of Aspergillus Conidia
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Round glass coverslips were washed in 70% (v/v) ethanol, coated with 0.1% (v/v) poly-L-lysine and air dried. The coverslips were then washed in deionized water and deposited in 12-well cell culture plates, which were blocked with 3% (w/v) milk powder in PBS-T. Germinated A. flavus and A. parasiticus conidia (overnight incubation in RPMI medium at 37°C) were added to the wells, and the plates were centrifuged (2,000 g, 15 min, room temperature) to deposit the germlings onto the coverslips. For direct staining, mAb AP3 (2 μg/ml) was added to the wells and incubated for 2 h at room temperature. After washing with PBS-T, AP3 binding to conidia and short hyphae was detected by adding 1.5 μg/ml GAMDylight 594 H+L (Jackson ImmunoResearch Laboratories) and incubating for 1 h at room temperature. The round coverslips were then placed upside down on a slide and sealed with nail polish to prevent desiccation. Samples were analyzed with a Leica DMR fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using excitation/emission maxima of 592/617 nm. A. fumigatus D141, ΔglfA and P. chrysogenum were detected with mAb AP3 and suitable Cy3-labeled secondary antibodies (Jackson ImmunoResearch Laboratories). Images were captured using a Leica SP-5 confocal laser scanning microscope (Leica Microsystems) as previously described (Wiedemann et al., 2016 (link)).
8
Imaging Golgi Localization of Gαo Variants
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For microscopy, N2a cells were transfected for 7 hours, trypsinized, and seeded on poly-l -lysine–coated coverslips in complete MEM for an additional 15 hours before fixation. Cells were fixed for 20 min with 4% paraformaldehyde in PBS, were permeabilized for 1 min using ice-cold PBS supplemented with 0.1% Triton X-100, blocked for 30 min with PBS supplemented with 1% BSA, incubated with the primary antibody against GM130 (dilution, 1:500; BD Biosciences, 610823) in blocking buffer for 2 hours at room temperature, washed, and subsequently incubated with the secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI) in blocking buffer for 2 hours at room temperature. The Cy3-labeled secondary antibody was from Jackson ImmunoResearch (dilution, 1:1000; 115-165-146). Coverslips were lastly mounted with VECTASHIELD on microscope slides. Cells were recorded with a Plan-Apochromat 63×/1.4 oil objective on a LSM 800 confocal microscope and further processed using the ZEN Blue software (all Zeiss). Quantification of relative localization of different variants of Gαo at plasma membrane and Golgi was performed as in (20 (link)).
9
Visualizing D2HGDH Localization in HEK-293 Cells
10
Immunofluorescence Imaging of Autophagy and Proliferation
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MSCs were seeded into 12-well plates with lysine-coated slides and stimulated with TNF (20 ng/ml, R&D Systems, 410-MT), IFNG (50 ng/ml, R&D Systems, 485-MI), or starved with minimum essential medium with Earle’s balanced salts (MEM/EBSS, Hyclone, SH30024.02) for 4 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich, T9284), blocked with 2% BSA (Santa Cruz, sc-2323), and incubated with anti-MAP1LC3 antibody (Cell Signaling Technology, 4108) overnight. Cells were washed with PBST and incubated with Cy3-labeled secondary antibody (Jackson, 111-165-003) and DAPI. For the MKI67 assay, mice were euthanized. Spinal cord and spleen were separated from naive mice and PBS-, shNC-MSC-, or shBecn1-MSC-treated EAE mice, and embedded in OCT. Frozen sections were cut and permeabilized with 0.1% (vol/vol) Triton X-100, blocked with 2% BSA, and stained with anti-MKI67 antibody (Abcam, ab15580) and anti-CD4 antibody (BD Biosciences, 550280). Cells were washed with PBST, and incubated with either Alexa 488- or Cy3-labeled secondary antibody (Invitrogen, CA11070), together with DAPI. Confocal microscopy (TCS SP5, Leica Microsystems, Wetzlar, Germany) was used for examining the slides and images were obtained.
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