Raw264.7 cells and BMDMs were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization for 10 min. After blocking with 5% BSA, the cells were incubated with primary antibodies against iNOS (1 : 200, ab178945, Abcam), Arg1 (1 : 50, #93668S, Cell Signaling Technology), and F4/80 (1 : 50, MAB5580-SP, R&D systems, USA) overnight at 4°C. The next day, corresponding secondary antibodies (1 : 500, Alexa Fluor® 488 goat anti-rabbit IgG and Alexa Fluor® 594 goat anti-mouse IgG, Abcam) were applied, followed by the nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI). Confocal lazer scanning analysis of Raw264.7 cells and BMDMs was performed using a laser confocal microscope (Zeiss LSM880, Carl Zeiss AG, Germany).
Alexa fluor 594 goat anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom, United States
Alexa Fluor 594 goat anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to bind to mouse immunoglobulin G (IgG) for detection and visualization purposes in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Abcam (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ab178945, by Abcam (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Mab5580 sp, by R&D Systems (1 mentions)
Goat anti chicken igy alexa fluor 488, by Abcam (1 mentions)
Donkey anti goat igg cy3, by Abcam (1 mentions)
Tcs sp5, by Leica Microsystems (1 mentions)
Hoechst 33258, by Beyotime (1 mentions)
Ae1 ae3, by Santa Cruz Biotechnology (1 mentions)
Monoclonal mouse anti n cad, by BD (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pet membrane, by Corning (1 mentions)
Anti myogenin f5d, by Santa Cruz Biotechnology (1 mentions)
Cd144 ve cadherin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Nanog, by ReproCELL (1 mentions)
Anti cd90, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Tcs sp2 confocal microscope, by Zeiss (1 mentions)
17 protocols using alexa fluor 594 goat anti mouse igg
1
Immunofluorescence Staining of Macrophages
2
Immunostaining of Fibroblasts and Schwann Cells
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Immunocytochemical staining was performed after differential digestion and differential adherence of Fbs and SCs. Fbs were fixed with 4% paraformaldehyde, blocked with blocking buffer (0.01 M PBS containing 5% goat serum) for 45 min at 37°C after washing, incubated overnight at 4°C with primary antibodies, including mouse monoclonal anti-CD90 (1:1000, Cat# ab225, Abcam) and goat anti-activin A (10 μg/mL, Cat# A1594-0.1MG, Sigma-Aldrich), and reacted with the following secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG (1:400, Cat# ab150116, Abcam) (Figure 1 ) and 488 goat anti-mouse IgG (1:400, Cat# ab150113, Abcam) and Cy3 donkey anti-goat IgG (1:400, Cat# ab6949, Abcam). Finally, the Fbs were subject to Hoechst 33258 (1:500, Cat# C1011, Beyotime Biotechnology) counterstaining at room temperature for 15 min.
After washing, SCs were fixed and blocked with blocking buffer (0.1% Triton X-100 in 0.01M PBS containing 5% goat serum), incubated overnight at 4°C with chicken anti-glial fibrillary acidic protein (anti-GFAP) primary antibody (1:500, Cat# ab254083, Abcam), and then incubated with Alexa Fluor 488 goat anti-chicken IgY (1:400, Cat# ab150169, Abcam). The SCs were then subject to Hoechst 33258 (1:500) counterstaining at room temperature for 15 min. Images were captured using a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany).
After washing, SCs were fixed and blocked with blocking buffer (0.1% Triton X-100 in 0.01M PBS containing 5% goat serum), incubated overnight at 4°C with chicken anti-glial fibrillary acidic protein (anti-GFAP) primary antibody (1:500, Cat# ab254083, Abcam), and then incubated with Alexa Fluor 488 goat anti-chicken IgY (1:400, Cat# ab150169, Abcam). The SCs were then subject to Hoechst 33258 (1:500) counterstaining at room temperature for 15 min. Images were captured using a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany).
3
Whole-Mount Immunohistochemistry Protocol
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Immunohistochemistry on whole-mount embryos was performed as described in54 using as primary antibodies an anti-BrdU (mouse, BD Biosciences, 1:100), a Zn-8 (mouse, ZIRC, 1:100) or a pan-cytokeratin (AE1/AE3) (mouse, Santa Cruz, 1:200) and as secondary antibody an Alexa Fluor® 594 (goat anti-mouse IgG, Abcam, 1:200).
4
Immunostaining and Western Blot Antibodies
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For immunostaining, the following primary antibodies were applied: polyclonal rabbit anti-DSG2 (#610121, Progen) and monoclonal mouse anti-N-cad (#610921, BD Transduction). As secondary antibodies, Cy2- and Cy3-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Dianova, Hamburg, Germany), STAR red goat anti-rabbit IgG (Abberior GmbH, Göttingen, Germany, #2-0012-011-9) or Alexa Fluor 594 goat anti-Mouse IgG (Abcam, Berlin, Germany, #ab150116) for STED were applied. For western blots, monoclonal mouse anti-Dsg1/2 (#61002, Progen, monoclonal mouse anti-N-cad (#610921, BD Transduction), mouse monoclonal anti-PG (#61005. Progen) and monoclonal mouse anti-Tubulin (#ab7291) primary antibodies were used. Polyclonal goat anti-rabbit (#111-035-045, Jackson ImmunoResearch Europe Ltd.) and mouse (#115-035-068, Jackson ImmunoResearch Europe Ltd.) HRP conjugated antibodies were used as secondary bodies.
5
Immunofluorescence Staining of Brain Tissue
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After anesthetized with pentobarbital sodium, the rats were perfused with PBS buffer solution and 4% paraformaldehyde transcardially, as described previously [30 (link),31 (link)]. Once removed, the brains were successively postfixed, embedded and cut into slices. After deparaffinized, sections were microwaved in citrate buffer for antigen retrieval and incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-3-NT antibody (Abcam, Cambridge, Massachusetts), rabbit polyclonal anti-MMP-9 antibody (Santa Cruz, California, USA), and rabbit polyclonal anti-CD68 (Abcam). After incubation with fluorescent conjugated secondary antibodies (Alexa Fluor 594 goat anti-mouse IgG, 1:500, Abcam; Alexa Fluor 488 goat anti-rabbit IgG, 1:500, Abcam) under room temperature for 1 h, the sections were restained by Hoechst 33258 for 10 min. The images were obtained by fluorescent microscopy (Bx51; Olympus Corporation, Shinjuku-ku, Japan).
6
CFTR Protein Detection in Lung Epithelial Cells
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CFBE41o− and 16HBE14o- were plated on a cell culture insert (0.75 × 106 cells per insert) containing a PET membrane (0.4 μm pore size) (www.corning.com ) to provide an air-liquid interface. Cells were transfected 12 h after plating with 5000 ng cmRNAhCFTR or equivalent (in nmol) pDNAhCFTR using Lipofectamine 2000 (www.invitrogen.com ) according to manufacturer’s instructions. Membranes were cut out from the insert 24 h after transfection, fixed with 4% PFA, blocked with 0.1% BSA and Fc blocker. Blocking was followed by overnight incubation with hCFTR clone 596 (1:250, kindly provided by the cystic fibrosis foundation therapeutics Inc.). As secondary antibody served Alexa Fluor 594 goat anti-mouse IgG (1:250, www.abcam.com , (ab150116)). Membranes were mounted on a coverslip and images were acquired by Zeiss Confocal Laser Scanning Microscope (CLSM) 710 NLO with Zen software.
7
Immunocytochemistry for Cell Characterization
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For immunocytochemistry, cells were cross-linked with 4% paraformaldehyde (PFA) for 15 min at room temperature. After washing with phosphate-buffered saline (PBS), cells were blocked with 4% normal goat serum/0.1% Tween 20/PBS for 1 h at room temperature and then incubated with primary antibodies. Primary antibodies used in this study were: anti-Pax7 (DSHB, Iowa City, IA, USA, 1:100), anti-MyoD (5.5A; Santa Cruz, Carlsbad, CA, USA, 1:100), anti-Myogenin (F5D; Santa Cruz, 1:200), anti-Myosin Heavy Chain (MF20; eBioscience, San Diego, CA, USA, 1:100), anti-GFP (MBL, Nagoya, Aichi, Japan, 1:200), CD144 (VE-cadherin; 1:100, eBioscience), and Nanog (Reprocell, Yokohama, Kanagawa, Japan, 1:100). The secondary antibodies used in this study were: Alexa Fluor 488 or Alexa Fluor 594 goat anti-mouse IgG, and Alexa Fluor 488 goat anti-rabbit IgG (Abcam, Cambridge, UK), all used at a 1:400 dilution. DAPI in anti-fading reagent was used for nuclear staining.
8
Identifying Cell Types in Tissue Culture
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Cultured DRG neurons, Schwann cells, and fibroblasts were first fixed in 4% paraformaldehyde (Sigma-Aldrich) for 30 minutes. They were then incubated with bovine serum albumin (5%; from Invitrogen) for 1 hour at room temperature to block nonspecific binding. Then, the cells were incubated with antibodies to the fibroblast marker Thy-1 cell surface antigen (anti-CD90) (mouse, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab225, RRID: AB_2203300), the Schwann cell marker S100 calcium-binding protein (Anti-S100) (rabbit, 1:1000, Sigma, Cat# SAB5500172) and the DRG neuron marker β3-tubulin (rabbit, 1:1000, Cell Signaling, Danvers, MA, USA, Cat# 5568S) at 4°C for 12–16 hours. Then, the cells were incubated with Alexa Fluor 594 goat anti-mouse IgG (1:400, Abcam, Cat# ab150116, RRID: AB_2650601) and goat anti-rabbit IgG H&L Alexa Fluor 488 (1:500, Abcam, Cat# ab150077, RRID: AB_2630356) secondary antibodies for 2 hours at room temperature. Nuclei were labeled with Hoechst 33342 (1:1000, Abcam, Cat# ab145597). The morphology of Schwann cells and fibroblasts was observed under a TCS SP2 confocal microscope and a Zeiss-ax10 fluorescence microscope (Carl Zeiss). Cell type and axon length statistics were confirmed using ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012).
9
Immunofluorescence Assay for Colon Tissue
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The immunofluorescence assay was performed following a previously described methods (Muñoz-Lorente et al., 2019) (link). For colon tissues, paraffin-embedded colon sections (5 μm) were deparaffinized in xylene and rehydrated in an ethanol gradient. Then, the colon pieces were treated with antigen retrieval solution (Biyuntian) at 95°C for 30 min. The sections were permeabilized with 0.5% Triton and then blocked with 1% BSA and 2% fetal bovine serum in PBS for 60 min. After blocking, the primary antibodies were subjected overnight in antibody diluents including anti-SCF rabbit antibody (cat. no. ab64677; Abcam), and anti-c-kit mouse antibody (cat. no. sc-365504; Santa Cruz Biotechnology). All primary antibodies were diluted 1:300 for the experiments. All sections were incubated for 1 h with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Abcam; both diluted 1:200) followed by incubation with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Immunofluorescence images were obtained using a confocal laser scanning microscope (BM-19AY, Shanghai BM Optical Instruments Manufacture Co. Ltd.).
10
Immunohistochemistry of Sheep Lung Tissue
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Immunohistochemistry was performed on frozen tissue of sheep lung tissue. Tissue sections were stained using previously described methods. Primary antibodies used were: anti-human KCNN4/K Ca 3.1 (rabbit polyclonal, 1:200; Lifespan Biosciences, Seattle, WA), anti-a-smooth muscle actin (a-SMA; mouse monoclonal, 1:800; Sigma-Aldrich), anti-human Ki-67 antibody (mouse polyclonal, 1:100; DAKO, Santa Clara, CA). For fluorescent staining, secondaries used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (both Abcam, Cambridge, UK). For peroxidase staining, goat antirabbit or mouse IgG (both Abcam) were used.
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