Antibodies to APP (6E10, Covance, Madison, WI, USA), cofilin (D3F9, Cell Signaling, Danvers, MA, USA), phospho-cofilin (77G2, Cell Signaling), actin (AC-74, Sigma Aldrich, St. Louis, MO, USA), tubulin (TU-02, Santa Cruz Biotechnology, Dallas, TX, USA), Aβ (D54D2, Cell Signaling), GFAP (Invitrogen), synapsin I (Invitrogen), PSD95 (Abcam, Cambridge, MA, USA), Drebrin (Abcam), microtubule-associated protein 2 (Millipore, Billerica, MA, USA), HRP-linked secondary antibodies (Jackson Immunochemicals, West Grove, PA, USA), and fluorescently labeled secondary antibodies (Invitrogen) were obtained from the indicated sources. The mouse monoclonal anti-RanBP9 antibody was a generous gift from Professor Elizabetta Bianchi (Pasteur Institute, France). Synthetic Aβ1-42 peptide was purchased from American Peptide (Sunnyvale, CA, USA). Aβ1-42 oligomers were prepared as previously characterized.21 (link) Briefly, Aβ1-42 powder was dissolved in hexafluoro-2-propanol (HFIP) at 1 mM for 30 min at room temperature, aliquoted to eppendorf tubes, allowed to evaporate overnight in fume hood, and subjected to speed vacuum for 1 h to remove traces of HFIP or moisture. To prepare Aβ oligomers, Aβ1-42 film was then dissolved in dimethyl sulfoxide (5 mM), and F-12 cell culture medium (without phenol) was added to a final concentration of 100 μM Aβ1-42 and incubated at 4 °C for 24 h.
Fluorescently labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescently labeled secondary antibodies are immunological reagents used in a variety of research and diagnostic applications. They are designed to bind to the Fc region of primary antibodies, allowing for the detection and visualization of target molecules or structures.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Lsm 510, by Zeiss (6 mentions)
Tissue tek 4583, by Sakura Finetek (3 mentions)
Hoechst 33258, by Thermo Fisher Scientific (3 mentions)
Sp5 fluorescence microscope, by Leica (2 mentions)
Dako target retrieval solution, by Agilent Technologies (2 mentions)
Millipore blocking reagent, by Merck Group (2 mentions)
Click it edu imaging kit with alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Ab15580, by Abcam (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Lsm 880 meta, by Zeiss (2 mentions)
Vectashield hardset antifade mounting medium with dapi, by Vector Laboratories (2 mentions)
Psd95, by Fujifilm (2 mentions)
Mab1596, by Bio-Rad (2 mentions)
76 protocols using fluorescently labeled secondary antibody
1
Preparation and Characterization of Aβ Oligomers
2
Immunofluorescence Staining of Vascular Cells
3
Embryo Immunofluorescence Staining Protocol
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Embryos were dissected and fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated by sucrose series, then frozen in a Tissue Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and processed to generate 10 μm frozen sections. For immunofluorescence staining, sections were treated with frozen absolute acetone for 10 min, air dried, washed with PBS and blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories) in 5% BSA/PBS for 15 min at room temperature. After incubation overnight at 4 °C with primary antibodies listed in Additional file 4 : Table S1, fluorescently labeled secondary antibodies (Invitrogen) were then used and incubated for 1 h at room temperature. After antibody staining, sections were counterstained with DAPI and mounted with fluorescent mounting medium (Dako). Imaging was performed on a Nikon Eclipse 80i.
4
Immunofluorescence Staining of Formalin-fixed Tissues
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Formalin fixed tissue sections (5 μm) were deparaffinized and rehydrated. After antigen retrieval with 10 mM sodium citrate buffer (pH 6.0) and blocking with 2% normal goat serum, specimens were incubated with antibodies specific for S. Typhimurium (Clone B395M, Dunn Laboratories, Asbach, Germany), CD3 (Abcam, Cambridge, UK), CD68 (Abcam, Cambridge, UK), myeloperoxidase (MPO) (Thermo Fisher Scientific, Schwerte, Germany), and MUC2 (Santa Cruz, Dallas, TX, USA) followed by fluorescently labeled secondary antibodies (Molecular Probes, Invitrogen, Carlsbad, CA, USA) or with fluorescently labelled DBA (dolichus biflorus agglutinin) and WGA (wheat germ agglutinin) lectins (Vector laboratories, Burlingame, CA, USA). Counterstaining of nuclei was performed using 4,6-Diamidin-2-phenylindol (DAPI) (Invitrogen, Carlsbad, CA, USA). Images were obtained using a Leica SP5 confocal microscope (Leica, Wetzlar, Germany).
5
Antibody-Based Protein Signaling Analysis
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The following antibodies were used: anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-phospho-Src family (Tyr416), anti-Yes, anti-Akt, anti-phospho-Akt (Ser473) and anti-p130Cas-pY410 from Cell Signalling Technology (Beverly, MA, USA); anti cortactin p80/85 (clone 4F11) and anti-phospho-cortactin (Tyr421) from Millipore (Temecula, CA USA); anti-β-catenin antibody (clone 6F9) and the monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) from Sigma-Aldrich (St. Louis, MO, USA); anti-E-cadherin and anti-p130Cas from BD Biosciences (Le Pont de Claix, France); anti-ERK1 (C-16) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Src and anti-Yes from R&D Systems (Abingdon, UK). Secondary antibodies coupled to horseradish peroxidase were from Jackson Immunoresearch Labs (West Grove, PA, USA). Fluorescently-labeled secondary antibodies were purchased from Invitrogen (Cergy Pointoise, France).
6
Immunofluorescence Staining of BBLF1, BGLF2, and Viral Proteins
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Cells were fixed with 4% paraformaldehyde and processed for indirect immunofluorescence staining, which was conducted using a primary antibody against Flag (Sigma), BBLF1 (Chiu et al., 2012 (link)), or BGLF2, following incubation with fluorescently labeled secondary antibodies (Invitrogen). Mouse anti-BGLF2 monoclonal antibody was generated using the synthesized peptide 145KTVEELQDITPS155 (Abmart, China). To colocalize BGLF2 with EA-D or gp350, cells were first stained with anti-BGLF2 mAb, then incubated with fluorescently labeled secondary antibody, and finally incubated with fluorescently labeled anti-EA-D (Millipore) or anti-gp350 (Millipore) that had been labeled using a Zenon mouse IgG labeling kit (Invitrogen). Images were captured using a Leica SP5 fluorescence microscope.
7
Immunofluorescence Labeling of Adipose Tissue
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Adipose tissues were fixed in 10% formalin and embedded in paraffin. 5 μm sections were prepared and stained with primary antibodies listed in Supplementary Table 2 . Briefly, sections were deparaffinized and rehydrated, followed by an antigen retrieval step in a modified citrate buffer (Dako Target Retrieval Solution, pH 6.1, Agilent). Sections were then incubated in Sudan Black (0.3% in 70% ethanol) to reduce the autofluorescence signal. Blocking was performed in Millipore blocking reagent (EMD Millipore), followed by incubating the section in primary antibody overnight at 4°C (Supplementary Table 2 ). The next days, slides were washed in PBST (0.1% Tween 20 in PBS) and were incubated with appropriate fluorescently labeled secondary antibodies (Invitrogen) at 1:200 dilution (Supplementary Table 2 ).
Detection of EdU labeling on slides was performed using Click-iT™ EdU Imaging Kit with Alexa Fluor™ 488 (ThermoFisher Scientific C10086) according to the manufacturer instructions. The EdU detection step preceded the antigen retrieval and staining with the antibodies.
The slides were mounted in mounting media with DAPI. Images were collected on a Zeiss LSM 710 NLO confocal microscope and processed with ImageJ.
Detection of EdU labeling on slides was performed using Click-iT™ EdU Imaging Kit with Alexa Fluor™ 488 (ThermoFisher Scientific C10086) according to the manufacturer instructions. The EdU detection step preceded the antigen retrieval and staining with the antibodies.
The slides were mounted in mounting media with DAPI. Images were collected on a Zeiss LSM 710 NLO confocal microscope and processed with ImageJ.
8
Isolation and Analysis of Colonic Lamina Propria Cells
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Isolation of colonic lamina propria cells was achieved using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Leukocyte isolation was performed with 40% / 80% discontinuous Percoll gradient (GE Healthcare). Cells were incubated with FcγR blocking reagent (rat anti-mouse CD16/CD32, BD Biosciences) for 30 minutes on ice prior to incubation with the other antibodies. Antibodies used for flow cytometry analysis are listed in S1 Table . To detect expression of Std fimbriae, flow cytometry was performed as previously described [16 (link)] with modifications. In brief, approximately 5x108 bacteria were fixed with 10% formalin and incubated at room temperature for 20 minutes. After washing with PBS, cells were resuspended in 2% NGS diluted in PBS and incubated at room temperature for 30 minutes. Polyclonal rabbit anti-StdA serum [38 (link)] was added to the cell suspensions following incubation at room temperature for 30 min. After washing with PBS, fluorescently labeled secondary antibodies (Invitrogen) were added. Flow cytometry was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec). The data were analyzed using FlowJo v.10 software (TreeStar).
9
Histological Analysis of Cartilage Tissue
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Following fixation, the samples were washed with PBS (Life Technologies), decalcified for 4 days in Versenate EDTA solution (American Master Tech). Processed samples were then taken through a sucrose gradient (10, 20, 30%), embedded in OCT compound (Tissue-Tek), sectioned (16 µm thick) on a cryotome (Leica), and mounted on glass slides (n = 4 section per sample). Antigen retrieval was performed with 1 mg/ml chondroitinase (Sigma-Aldrich) and 5 mg/ml hyaluronidase (Sigma-Aldrich) for 30 min at 37°C. Nonspecific binding was suppressed with 1% horse serum (Vector Labs) in PBS for 45 min. Slides were then washed with 0.1% Triton X-100/TBS, blocked in 1% BSA, incubated with primary antibodies against collagen type II (Col2) (Abcam), myosin heavy chain (MHC) (Developmental Studies Hybridoma Bank), and/or proliferating cell nuclear antigen (PCNA) (Abcam) overnight at 4°C, and incubated with fluorescently labeled secondary antibodies (Invitrogen) for 1 h at room temperature. Samples were counterstained with DAPI (Invitrogen) and imaged with an Olympus CKX41 microscope outfitted with a Leica DFC 3200 camera.
10
Integrin-Targeted Immunohistochemistry Protocol
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All antibodies were obtained from Cell Signaling Technology (CST, Danvers, MA, USA) unless indicated. The antibodies for integrin αv, integrin α7, integrin β1, integrin β3, and EGFR (wild type, for immunohistochemistry) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For IHC staining only, anti-EGFRvIII specific antibody was from Biosynthesis Biotechnology Co. Ltd., Beijing, China; others from ZSGB-Bio Co. Ltd., Beijing, China. Fluorescently labeled secondary antibodies and phalloidin were from Invitrogen (Courtaboeuf, France). Plasma vitronectin were from Sigma-Aldrich (StLouis, MO, USA). The integrin αvβ3 and αvβ5 antagonist cilengitide (Selleck Chemicals Co.Ltd, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −20°C. Cell culture medium was used to serially dilute stock solutions to the required concentrations.
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