General cell morphology was visualized after fixation with 4% paraformaldehyde through staining with 4% Giemsa solution for 1 min, washing with distilled water and natural drying. For cytoskeleton β-actin immunocytochemistry, after fixation with 4% paraformaldehyde, cells were permeabilized with 100% methanol at −20 °C for 15 min, and 0.5% Triton X-100 (Amresco Inc., Solon, OH, USA) in PBS for 15 min. Non-specific binding was blocked with 1% goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. After each incubation step, the samples were washed thrice under gentle agitation on a horizontal shaker table for 5 min. The primary antibodies anti β-actin (mouse monoclonal anti βACT antibody, working dilution of 1:1000, MA1-140, Thermo Fisher Scientific, Rockford, IL, USA) were incubated overnight under refrigeration and were followed by corresponding goat anti-mouse (IgG FITC, working dilution of 1:1000, Sigma Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Hoechst’s solution (H3569, 2μL/mL for 5 min, Invitrogen, Eugene, OR, USA) was used for nuclear staining. Cultures were examined under a phase contrast microscope (Zeiss Axio Vert.A1, Zeiss, Jena, Thuringia Land, Germany), equipped with an AxioCam MRC camera (Zeiss, Jena, Thuringia Land, Germany).
Goat anti mouse igg fitc
Manufactured by Merck Group
Sourced in United States
Goat anti-mouse IgG-FITC is a laboratory reagent used in various immunoassays and cell-based applications. It is a conjugate of goat-derived polyclonal antibodies specific to mouse immunoglobulin G (IgG) and the fluorescent dye fluorescein isothiocyanate (FITC). This product can be utilized for the detection and visualization of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Immobilon membrane, by Merck Group (2 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (2 mentions)
Rabbit anti iκβα antibody, by Merck Group (2 mentions)
Goat anti mouse igg peroxidase, by Merck Group (2 mentions)
Ecl plus substrate, by CWBIO (2 mentions)
Rabbit anti ikkα antibody, by Merck Group (2 mentions)
Goat anti rabbit igg fitc, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Ripa lysis buffer, by CWBIO (2 mentions)
Anti αvβ3, by Abcam (2 mentions)
Anti αvβ5, by Merck Group (2 mentions)
Anti α5 fitc, by BioLegend (2 mentions)
Anti β1 af488, by BioLegend (2 mentions)
Guava easycyte flow cytometer, by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Axio vert a1, by Zeiss (1 mentions)
31 protocols using goat anti mouse igg fitc
1
Cell Morphology and Cytoskeleton Visualization
2
Investigating Anti-Cancer Mechanisms
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All cell culture media, antibiotics, and trypsin were purchased from Gibco (Grand Island, NY, USA), and foetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Rabbit anti-NF-κΒ p65 antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-caspase-8 antibody, rabbit anti-GAPDH antibody, rabbit anti-Poly[ADP-ribose] Polymerase (PARP) antibody, rabbit anti-cyclin B1 antibody, rabbit anti-survivin antibody, rabbit anti-caspase-3 antibody, mouse anti-α-tubulin antibody, rabbit anti-β-actin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, DAPI, methyl thiazolyl tetrazolium (MTT), and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Immobilon membranes were purchased from Merck Millipore (Bedford, MA, USA). Cell Tracker CM-Dil and Calcein-AM were purchased from Invitrogen (Carlsbad, CA, USA). ECL Plus substrate, bicinchoninic acid (BCA) reagents, and RIPA lysis buffer were purchased from CWBio (Beijing, China). Kanglaite injection was purchased from Zhejiang Kanglaite Pharmaceutical Co., Ltd, (Zhejiang, China).
3
Integrin Expression Profiling in MB-004 Cells
4
Immunofluorescence Characterization of hESC-RPE Cells
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RPE markers were also analyzed in hESC-RPE using immunofluorescence. For this, passage 2 RPE cells were fixed with 4% paraformaldehyde for 15 min, permea-bilized with 0.1% Triton X-100, and blocked with a solution containing 2% goat serum (Normal Goat Serum - NGS) 0.5% BSA, and 0.1% Triton X-100 diluted in PBS for 1 hour. Primary antibodies were diluted in 2% NGS / PBS-T 0.3% and incubated at 4℃ overnight, using the following dilutions: 1：50 of the rabbit polyclonal transcription factor associated with microphthalmia, MITF (Abcam); 1：250 of mouse monoclonal bestrofin membrane protein, BEST (Pierce). Goat anti-mouse IgG FITC (1：32; Sigma), and goat anti-rabbit IgG rhodamine (Jackson ImmunoResearch Laboratories) were diluted in blocking solution and incubated with samples for 1 hour at room temperature. Nuclear staining was executed using 0.2 μg/ml DAPI (Sigma) diluted in PBS. The slides were assembled using Vectashield (Vector Laboratories). The images were obtained using a confocal microscope (Confocal Zeiss 5 LIVE).
5
Quantifying Epithelial-Mesenchymal Transition
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Cells (3×104) were seeded on coverslips for 24 h, the invasive/stemness phenotype was experimentally induced as explained above, after which cells were fixed with paraformaldehyde 4% for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 20 min. Cells were blocked for 1 h and then stained overnight at 4°C with the primary antibodies: mouse monoclonal anti-E-cadherin (1:100, Clone: 36/E-cadherin, BD Biosciences. ref. 610181), rabbit monoclonal anti-vimentin-Alexa Fluor-594 (1:1000, Clone: EPR3776. ref. ab154207), rabbit polyclonal anti-Oct4 (1:100, ref. ab18976) or rabbit polyclonal anti-Sox2 (1:100, ref. ab97959); all antibodies were from Abcam. After that, cells were incubated for 30 min with the secondary antibodies: goat anti-mouse-IgG-FITC (1:500 Sigma-Aldrich Co., ref. F0257) or donkey anti-rabbit-FITC (1:500, Jackson ImmunoResearch Laboratories, ref. 711-095-152). Finally, nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; Thermo Fisher Scientific, ref. D1306) for 25 min. Cells were observed using a fluorescence microscope Olympus BX51 and images were acquired with a digital camera (Camedia C4040, Olympus). FITC staining intensity was quantified using the Image Pro Plus software, and the integrated optical density (IOD) of green cells was obtained.
6
Immunohistochemistry Analysis of Neurite Outgrowth
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Immunohistochemistry techniques were performed as previously described [70 (link)]. Briefly, cells were fixed in 4% paraformaldehyde at room temperature for 30 min. To block nonspecific antibody binding, samples were incubated in 2% goat serum/2.5% BSA/0.05% Triton-X-100 in 1× PBS for 30 min. Primary antibody (anti-β-Tubulin III diluted 1:1000, Sigma, St. Louis, MO, USA) was prepared in 10% goat serum/2.5% BSA/0.05% Triton-X-100/0.1% sodium azide in 1× PBS and incubated with cells overnight. Cells were then washed in 1× PBS and incubated in the secondary antibody Goat anti-mouse IgG (FITC) diluted 1:200 (Sigma) for 4 h. Prolong Gold (Molecular Probes/Invitrogen, Carlsbad, CA, USA), an antifade agent with 4′,6-diamidino-2-phenylindole (DAPI), was used to stain nuclei. The neurons were imaged using an EVOS fluorescent microscope and analyzed with Metamorph imaging software (Molecular Devices, LLC. Sunnyvale, CA, USA). Neurite number, total neurite growth, mean neurite growth, and cell body area were all recorded.
7
Immunostaining of HUVEC-WJ-MSC Bilayers
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HUVEC and WJ-MSC bilayers were fixed with 4% PFA at 2 and 24 h. Cells were permeabilized with 0.15% triton X-100, blocked with 5% goat serum for 30 min at RT, then incubated overnight at 4°C with mouse anti-human VE-cadherin (10 µg/ml) (BD Biosciences, U.K. catalog# 5556661). After washes, cells were incubated for 2 h in the dark with goat anti-mouse IgG-FITC (Sigma Aldrich, U.K., catalog# F0257) (1:100). Unbiased ten images per coverslip were obtained with Nikon fluorescence microscope with appropriate filters to allow visualization of PKH26 and FITC (duration of co-culture and sample ID were blinded). Images were used to count paracellular junctions and characterised according to VE-cadherin staining pattern at cell-cell borders: continuous or disrupted. A grid was used by ImageJ software so that all the junctional regions had the same chance of being counted, and junctions from every other square which did not cross the ‘forbidden line’ were analysed [23 (link),24 (link)].
8
BrdU Incorporation Assay for Cell Cycle
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Cells were treated with SH003 and DTX and 10 μM BrdU (Sigma, USA) was added to the cell culture medium for 1 h prior to harvest. BrdU-labeled cells were harvested, washed with 1 × HBSS (Thermo Fisher Scientific, MA, USA) to remove unincorporated BrdU, and fixed in 70% EtOH. The cells were denatured in 2 N HCl/0.5% Triton X-100 for 30 min at RT and neutralized by adding to 0.1 M sodium tetraborate (pH 8.5) for 2 min at RT. The cells were then resuspended in PBS containing with 0.5% Tween-20 and 1% BSA, incubated with anti-BrdU (1:50) (Santa Cruz Biotechnology, TX, USA) for 30 min at RT and stained with 10 µg of Goat anti-Mouse IgG-FITC (Sigma, USA) for 30 min. After washing, the cells were stained with 5 µg/mL PI solution containing sodium citrate, RNase A, and NP-40 in PBS for 30 min on ice. Cell cycle distribution and BrdU positive cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, USA). The results were analyzed using CellQuest Pro software version 5.2 (BD Biosciences, USA). S-phase arrest was represented as the proportion of BrdU-negative cells in S phase.
9
Integrin Expression Profiling in MB-004 Cells
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Integrin expression profiling was performed with 4×104 MB-004 cells per reaction. Cells were pelleted and resuspended in 50 μL of a 1:100 dilution of anti-αVβ3 (Abcam), 1:100 dilution of anti-αVβ5 (Millipore), 1:25 dilution of anti-α5-FITC, or 1:25 dilution of anti-β1-AF488 (BioLegend) antibodies. Cells were incubated with primary antibodies for 40 min on ice and were then washed in PBS containing 0.1% bovine serum albumin (PBS/BSA). Cells treated with unlabeled antibodies were then incubated with goat anti-mouse IgG-FITC (Sigma) for 40 min and washed in PBS/BSA. Cells were analyzed on a Guava EasyCyte flow cytometer (EMD Millipore), and the resulting data was analyzed using FlowJo software (TreeStar).
10
Quantification of Anti-Nuclear Antibodies
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