Briefly, the samples were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2–0.5% Triton X-100. The samples were then blocked for 30 min and incubated with various mouse or rabbit primary antibodies (Supplementary Table S3 ) for four hours. For surface markers, no permeabilization was performed. After washing, the cells were incubated with the corresponding secondary antibody: Alexa Fluor® 488 goat anti-Mouse IgG1, 488 or 594 goat anti-Rabbit IgG, or 594 donkey anti-Goat IgG (Life Technologies) for one hour. The samples were counterstained with Hoechst 33342 and visualized using a fluorescent microscope (Olympus IX70, Melville, NY). For 5-Bromo-2′-deoxyuridine (BrdU) assay, the cells were incubated in medium containing 10 µM BrdU (Sigma) for four hours. The cells were then fixed with 70% cold ethanol and denatured using 2N HCl/0.5% Triton X-100 for 30 min in the dark. The samples were reduced with 1 mg/mL sodium borohydride for 5 min and incubated with mouse anti-BrdU (1:100, Life Technologies) in blocking buffer (0.5% Tween 20/1% bovine serum albumin in PBS), followed by Alexa Fluor® 488 goat anti-Mouse IgG1. The cells were counterstained with Hoechst 33342 and analyzed by a fluorescent microscope and the ImageJ software.
Alexa fluor 488 goat anti mouse igg1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 goat anti-mouse IgG1 is a fluorescently labeled secondary antibody used for detection and visualization of mouse IgG1 primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (5 mentions)
Goat anti mouse igg2a alexa fluor 546, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Goat anti mouse igg2a alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Mouse monoclonal igg1 anti c myc 9e10 sc 40, by Santa Cruz Biotechnology (3 mentions)
Streptavidin apc, by Southern Biotech (3 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti mouse igg2a, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse igg2b alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Alexa fluor 568 goat anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Mouse igg1, by BD (2 mentions)
Zymoprep yeast plasmid miniprep 2 kit, by Zymo Research (2 mentions)
Lysozyme, by GeneTex (2 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (2 mentions)
Ba d5, by Developmental Studies Hybridoma Bank (2 mentions)
Bf f3, by Developmental Studies Hybridoma Bank (2 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
62 protocols using alexa fluor 488 goat anti mouse igg1
1
Immunofluorescence and BrdU Labeling Assay
2
Whole-Mount Immunofluorescence of Zebrafish Hearts
3
Flow Cytometry Analysis of Pluripotency Markers
4
Multi-Marker Immunophenotyping of Cell Populations
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An aliquot of 5 × 105 cells was incubated with 100 μl of primary monoclonal antibody mixture containing anti-keratin MNF116, IgG1 (DAKO) final concentration 3,2 μg/ml, anti-keratin AE1/AE3, IgG1 (Millipore–Chemicon), final concentration 10 μg/ml and anti-vimentin 3B4, IgG2a (DAKO, Glostrup, Denmark), final concentration 3,1 μg/ml for 30 min at 4 °C, in PBATw.
Cells were washed twice with ice-cold PBATw and centrifuged at 1,000 g for 5 min at 4 °C. Then cells were incubated with 100 μl premixed secondary reagents Alexa Fluor® 488 Goat Anti-Mouse IgG1 (Life Technologies) final concentration 2,5 μg/ml for keratin detection, Alexa Fluor® 647 Goat Anti-Mouse IgG2a (Life Technologies), final concentration 2,5 μg/ml for vimentin detection, in PBATw.
After 60 min at 4 °C, cells were washed twice with ice-cold PBATw and incubated, for 30 min at 37 °C, with 0,5 ml DNA staining solution containing 10 μM DAPI (Sigma-Aldrich) in PBATw. After incubation time sample was washed twice with PBATw by a 5 min centrifugation at 1,000 g and pellet was resuspended in the same buffer.
Cells were washed twice with ice-cold PBATw and centrifuged at 1,000 g for 5 min at 4 °C. Then cells were incubated with 100 μl premixed secondary reagents Alexa Fluor® 488 Goat Anti-Mouse IgG1 (Life Technologies) final concentration 2,5 μg/ml for keratin detection, Alexa Fluor® 647 Goat Anti-Mouse IgG2a (Life Technologies), final concentration 2,5 μg/ml for vimentin detection, in PBATw.
After 60 min at 4 °C, cells were washed twice with ice-cold PBATw and incubated, for 30 min at 37 °C, with 0,5 ml DNA staining solution containing 10 μM DAPI (Sigma-Aldrich) in PBATw. After incubation time sample was washed twice with PBATw by a 5 min centrifugation at 1,000 g and pellet was resuspended in the same buffer.
5
Immunofluorescence Staining of EC and SMC
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Passage 1 EC and SMC were grown to confluence on plastic dishes or on chitosan/PCL membranes. After that, they were fixed with 4% PFA (paraformaldehyde) for 10 min, permeabilized with 0.05% Triton X-100 for 10 min, and blocked with 1% BSA (bovine serum albumin). The cells were stained with primary antibodies overnight at 4 °C, washed with PBS and incubated with secondary antibodies for 1 h at room temperature. The stained cells were analysed with an inverted fluorescence microscope (Nikon Ti-E) using Nikon AR software.
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).
6
Immunofluorescence Staining of Cells
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Briefly, the samples were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2–0.5% Triton X-100. The samples were then blocked for 30 min and incubated with various mouse or rabbit primary antibodies (Supplementary Table S1 ) for four hours. For surface markers, no permeabilization was performed. After washing, the cells were incubated with the corresponding secondary antibody: Alexa Fluor® 488 goat anti-Mouse IgG1, Alexa Fluor® 488 or 594 goat anti-Rabbit IgG, or 594 donkey anti-goat IgG (Life Technologies) for one hour. The samples were counterstained with Hoechst 33342 and visualized using a fluorescent microscope (Olympus IX70, Melville, NY) or a confocal microscope (Zeiss LSM 880).
7
Yeast-Display Screening for Ligand Binding
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Yeast-display experiments were conducted essentially as described [3 (link)]. Briefly, yeast cells were grown in selective medium SDCAA overnight at 30°C. The cells were then resuspended in 10 ml induction medium and incubated at 20°C for 20 h. 107 cells were then used for yeast-cell surface display experiments: cells were subjected to primary antibody (mouse monoclonal IgG1 anti-c-Myc (9E10) sc-40, Santa Cruz Biotechnology) for expression monitoring and biotinylated ligand at 90 nM lysozyme (GeneTex) in PBS-F for 30 min at room temperature. The cells then underwent a second staining with fluorescently labeled secondary antibody (AlexaFluor488—goat-anti-mouse IgG1 (Life Technologies) for scFv labeling, Streptavidin-APC (SouthernBiotech) for ligand labeling) for 10 min at 4°C. Next, the cell fluorescence was measured and cells were collected under sorting conditions for expression and top 15% binders. The selection gates were calibrated using the wild-type scFv D44.1 and these gates were subsequently applied to the library constructs. Following fluorescence-activated cell sorting (FACS), cells were grown in SDCAA for 1–2 days and plasmids were extracted using Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research).
8
Immunohistochemical Antibody Protocol
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DHB was obtained from ProteoChem (Hurricane, HT, USA). 1,5-DAN, formic acid, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance LC–grade water (H2O), acetone, methanol, and acetonitrile (ACN) were purchased from Fisher Scientific (Fair Lawn, NJ). BAD5, BFF3, BF35, and SC71 antibodies were purchased as 1-ml supernatants from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA); cytochrome c antibody was purchased from Abcam (Cambridge, England). Secondary antibodies Alexa Fluor 488 Goat anti-Mouse IgG1, Alexa Fluor 568 Goat anti-Mouse IgM, and Alexa Fluor 647 Goat anti-Mouse Ig2b were purchased from Life Technologies (Grand Island, NY). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology (Shanghai, China).
9
Double Immunofluorescence for CD14 and CD163
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Three basalis samples and three parietalis samples with various CD163/CD14 ratios were randomly selected to perform double label immunofluorescence of CD14 and CD163. Paraffin‐embedded sections (4 μm) were deparaffinized for 3x5 min in xylol and hydrated via graded ethanol to demi‐water. Then sections were pre‐treated with heated citrate for 10 min and cooled down for 30 min, followed by washing in PBS for 5 min. Sections were incubated overnight with a mix of primary antibodies CD14 (rabbit anti‐human IgG, dilution 1:500, D7A2T, CellSignaling, USA) and CD163 (mouse anti‐human IgG1, dilution 1:20, 10D6, Abcam, UK). Thereafter, the sections were washed with PBS for 3x5 min, and incubated with a mix of secondary antibodies: Alexa Fluor 546 goat‐anti‐rabbit IgG (dilution 1:200, Life Technologies, the Netherlands) and Alexa Fluor 488 goat‐anti‐mouse IgG1 (dilution 1:200, Life Technologies). Then the sections were mounted with ProLong Gold antifade mount with 4′,6‐diamidino‐2‐phenylindole (DAPI) (70 μl/slide, Life Technologies), and stored in the dark at room temperature for approximate 2 h.
10
Immunofluorescence Assay for Biomarker Detection
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For biomarker detection, the cells were fixed using 4% paraformaldehyde (PFA) and permeabilized using 0.2% Trixton-X 100. The samples were blocked with 5% FBS in PBS and stained with the primary antibodies (Supplementary Table S1 ), followed by the corresponding anti-species Alexa Fluoro antibodies, i.e., Alexa Fluor 488 goat anti-mouse IgG1 or Alexa Fluor 594 goat anti-Rabbit IgG (Life technologies). Both primary and secondary antibody dilutions were made based on the manufacturer’s recommendations and prepared in staining buffer (2% FBS in PBS). Then the nuclei were counterstained with Hoechst 33342 (blue), and pictures were taken for blue, green, and red colors to detect the markers and their cellular locations under a fluorescent microscope (Olympus IX70, Melville, NY).
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