Mouse monoclonal anti-chikungunya virus E2 envelope glycoprotein antibodies, clone CHK-48 (produced in vitro) (NR-44002) were obtained from BEI Resources, NIAID, NIH. These antibodies were shown to be cross-reactive with MAYV antigens of both BE AN343102 and BE AR505411 strains. Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibodies, Alexa Fluor 488 (A-11029) were purchased from Invitrogen. Both antibodies were used in focus-forming assays at a dilution of 1:1000.
Alexa fluor 488 a 11029
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 (A-11029) is a fluorescent dye. It can be used to label and detect molecules in biological samples. The dye emits green fluorescence when excited by a suitable light source.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Clone chk 48, by BEI Resources (1 mentions)
Anti map2ab, by Merck Group (1 mentions)
Anti gfap, by BioLegend (1 mentions)
Anti gaba, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bz x800 analyzer software, by Keyence (1 mentions)
Alexa fluor 488 a 11034, by Thermo Fisher Scientific (1 mentions)
P mypt1, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 a11032, by Thermo Fisher Scientific (1 mentions)
Fusion software, by Vilber (1 mentions)
Anti c ebpβ sc 150, by Santa Cruz Biotechnology (1 mentions)
Anti cebpα 8178, by Cell Signaling Technology (1 mentions)
Anti pparγ sc 7273, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluorsave reagent, by Merck Group (1 mentions)
Ab76198, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
10 protocols using alexa fluor 488 a 11029
1
Cross-reactive Antibodies for Chikungunya and Mayaro Viruses
2
Immunostaining of Neural Cell Types
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Cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 15 min, then permeabilized in PBS containing 0.3% (w/v) Triton X-100 for 10 min, lastly blocked in PBS containing 4% (w/v) BSA for 2 h. Cells were incubated with the following primary antibodies (1:250 dilution) for 2 h at room temperature): anti-βIII-tub (Biolegend, cat. 801201), anti-MAP2ab (Merck Millipore, cat. AB5622), anti-GFAP (Biolegend, cat. 83721), and anti-GABA (Sigma Aldrich, cat. A2052). Subsequently, the cells were rinsed three times with 0.1% (w/v) BSA in PBS-Tr and incubated with the secondary antibody (1:500 dilution) for 1 h at room temperature in the dark (Alexa Fluor 488 A11029, and Alexa Fluor 568, A11036, Invitrogen). Nuclei were counterstained with DAPI (Invitrogen, cat. 1:10000) for 45 min. Cell images were acquired using a Keyence fluorescence microscope BZ-X710E equipped with the BZ-X800 Analyzer software (Keyence, Osaka, Japan) with a 20X Plan-Apo Gamma NA 0.75 objective and fluorescence filter set for GFP, TRITC, and DAPI.
3
Immunoblotting and Immunofluorescent Assay Protocol
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Immunoblotting and immunofluorescent assay were performed as described previously [40 (link)]. Antibody for CHCHD2 (#19,424-1-AP) was purchased from Proteintech. Antibodies for cleaved caspase-3 (C.Casp-3, #9664), OCT-4A (#2840), pPTEN (#9551), and pMYPT1 (#5613) were purchased from Cell Signaling Technology. Antibodies for α-tubulin (#sc-5286) and β-actin (#sc-47778) were purchased from Santa Cruz Biotechnology. Antibody for BCL-xL (#ab32370) and phosphor-BAX (#ab111391) were purchased from Abcam. Antibodies for Flag (#F1804) and active BAX (#MABC1176M) were purchased from Sigma-Aldrich. Quantification of blots was performed by Fusion software (Vilber Lourmat) in accordance with the manufacturer’s protocol. For immunofluorescent assay, secondary antibodies for mouse primary antibody conjugated to Alexa Fluor 488 (A11029) and Alexa Fluor 594 (A11032) fluorophores as well as for rabbit primary antibody conjugated to Alexa Fluor 488 (A11034) and Alexa Fluor 594 (A11037) were purchased from Invitrogen. Nucleus staining reagent 4’,6-diamidini-2-phenylindole (DAPI, #D1306) was purchased from Thermo Fisher Scientific. Fluorescence microscopy [Olympus, BX53 (Light source: 103W mercury lamp/12V 100W halogen lamp, Software: CellSense)] was used for imaging samples.
4
Immunodetection of Adipogenic Markers
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Anti‐laminin (L9393; 1:400) antibody was purchased from Sigma‐Aldrich and secondary antibody Alexa Fluor 488 (A11029; 1:800) was purchased from Invitrogen. Anti‐PDGFRα (#3174), anti‐perilipin (#9349), anti‐FABP4 (#3544) and anti‐C/EBPα (#8178) primary antibodies were purchased from Cell Signalling and used at 1:500 dilution. Anti‐PPARγ (sc‐7273) and anti‐C/EBPβ (sc‐150) primary antibodies were purchased from Santa Cruz and used at 1:200 dilution. Anti‐mouse (sc‐2005) and anti‐rabbit (sc‐2004) HRP‐conjugated secondary antibodies were purchased from Santa Cruz and used at 1:4000 dilution.
5
Immunofluorescence Staining of Polyglutamine
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Cells were fixed with 4% PFA in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After permeabilization, the cells were blocked with 3% BSA in 0.2% Triton X-100 in PBS for 10 min. The cells were incubated with mouse anti-polyQ (MAB1574, 1:50; Millipore) primary antibody for 2 h. After washing with 0.2% Triton-X in PBS, the cells were incubated with goat anti-mouse Alexa Fluor 488 (A-11029, 1:500; Thermo Fisher Scientific) secondary antibody and 2 μg/ml Hoechst 33342 (1656104; Life Technologies) for 1 h. The cells were washed with 0.2% Triton-X in PBS and with distilled water. The coverslips were mounted with FluorSave reagent (Merck Millipore).
6
Immunofluorescence Staining of Tissue Sections
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Subcutaneous and aortic tissue sections were first blocked with 1% (in PBS) bovine serum albumin (BSA, Sigma-Aldrich) solution for 30 min and then incubated with the following primary antibodies in 1% BSA solution overnight at 4 °C: CD11b (rabbit polyclonal IgG, PA5-90724, Thermo Fisher Scientific, USA), CD68 (rabbit polyclonal IgG, ab125212, Abcam, USA), CD206 (rabbit polyclonal IgG, ab64693, Abcam, USA), alpha-smooth muscle Actin (αSMA, mouse monoclonal IgG2a, ab7817, Abcam, USA), and endothelial nitric oxide synthase (eNOS, mouse monoclonal IgG1, ab76198, Abcam, USA). After that, samples were washed with PBS and incubated with goat anti-rabbit secondary antibody (Alexa Fluor 594, A-11037, Thermo Fisher Scientific, USA) or goat anti-mouse antibody (Alexa Fluor 488, A-11029, Thermo Fisher Scientific, USA) for 1 h at room temperature. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, Krackeler Scientific, USA). Tissue sections without primary antibody incubation were used as negative controls. The stained samples were imaged with the inverted immunofluorescence microscope (Eclipse Ti2, Nikon, Japan).
7
Immunostaining of Stemness and DNA Damage
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Spheroid cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following cell fixation, cells were incubated with antibodies recognizing CD133 (MBS462020; MyBioSource), Nanog (#4893; Cell Signaling), and γ-H2AX (05-636; Millipore) in a solution of PBS with 1% FBS and 0.1% Triton X-100 at 4 °C overnight. Cells were then stained with secondary antibodies tagged with Alexa Fluor 488 (A-11029) or Alexa Fluor 568 (A-11061), both from Thermo Fisher. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, 28718-90-3; Sigma-Aldrich). Stained cells were visualized on an inverted confocal microscope (Leica Microsystems) and images were processed using Imaris version 7.6 (Bitplane). Three samples were examined for each group, and each sample was visualized for five fields.
8
Immunofluorescence Microscopy with RNA Digestion
9
Western Blot and Immunocytochemistry Antibody Protocol
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The following antibodies were used for western blots as specified; 1:1000 FLAG-M2 (mouse, Sigma F1804), 1:1000 GAPDH 65C (mouse, Santa Cruz sc32233), 1:1000 tubulin (mouse, DSHB 12G10), 1:1000 PERK (rabbit, CST 3192S), 1:1000 phospho-eIF2α (rabbit, Thermo MA5-15133), 1:1000 BiP (rabbit, CST 3177S), 1:5000 puromycin 12D10 (mouse, Millipore MABE434), in 5% non-fat dry milk (NFDM). LI-COR IRDye 680RD goat-anti-mouse secondary antibody (96-68070) was used 1:10,000 in 5% NFDM for GAPDH and tubulin loading controls. HRP-conjugated goat-anti-mouse (115-035-146) or goat-anti-rabbit (111-035-144) antibodies from Jackson ImmunoResearch Laboratories were used at 1:10,000 in 5% NFDM for all other western blots. Full-length images of the western blots from Figs. 1 b and 3b, f are supplied in Supplementary Fig. 7 .
The following antibodies were used for immunocytochemistry as specified; 1:500 FLAG (rabbit, Cell Signaling #2368), 1:200 G3BP (mouse, BD Transduction Laboratories 23/G3BP), 1:200 FMRP (mouse, Covance 6B8), and 1:200 FMRP (rabbit, abcam17722) in 5% normal goat serum (NGS—HEK293 cells) or 2% bovine serum albumin (BSA—MEFs). Secondary goat-anti-mouse Alexa Fluor 488 (A-11029) and goat-anti-rabbit Alexa Fluor 555 (A-21428) from Life Technologies were applied at 1:500.
The following antibodies were used for immunocytochemistry as specified; 1:500 FLAG (rabbit, Cell Signaling #2368), 1:200 G3BP (mouse, BD Transduction Laboratories 23/G3BP), 1:200 FMRP (mouse, Covance 6B8), and 1:200 FMRP (rabbit, abcam17722) in 5% normal goat serum (NGS—HEK293 cells) or 2% bovine serum albumin (BSA—MEFs). Secondary goat-anti-mouse Alexa Fluor 488 (A-11029) and goat-anti-rabbit Alexa Fluor 555 (A-21428) from Life Technologies were applied at 1:500.
10
Immunofluorescence Imaging of p50/p105
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The p50/p105-Flp-In cell lines were seeded on coverslips coated with rattail collagen (Thermo Fisher Scientific). Tetracycline-treated cells were induced with TNF (10 or 25 ng/mL for 40 minutes), fixed in paraformaldehyde, and blocked with 1% BSA-PBS (Sigma-Aldrich). The HA.11 antibody was used for detection of tagged p50/p105 with anti-mouse Alexa Fluor 488 (A-11029, Life Technologies) secondary antibody. Nuclei were stained with 49-6-diamidino-2-phenylindole (D9542-5MG, Sigma-Aldrich). Imaging was performed at 340 magnification with the Axio Imager.Z2 (Zeiss, Oberkochen, Germany) and the Nuance multispectral imaging system FX (PerkinElmer, Waltham, Mass). ImageJ software (National Institutes of Health, Bethesda, Md) was used for quantification of signal intensities. 29 Three hundred to 600 cells per condition were analyzed.
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