Single‐cell suspensions of placental leukocytes were prepared, as previously described (Shields et al. 2018 ). Briefly, one placenta from each rat was homogenized and filtered through a 70‐μm cell strainer and resuspended in 15 mL of Rosswell Park Memorial Institute medium (RPMI) (10% FBS). Whole blood was collected in an EDTA tube and diluted with 5 mL of RPMI. Peripheral blood mononuclear cells (PBMCs) and placental lymphocytes were isolated by centrifugation on a cushion of Ficoll‐Isopaque (Lymphoprep, Accurate Chemical & Scientific Corp., Westbury, NY) according to the instructions of the manufacturer. Single‐cell suspensions (1 × 106 cells) were stained for flow cytometry after blocking with 10% goat and mouse serum. Antibodies used for flow cytometry were as follows: VioGreen anti‐CD3 (Miltenyi Biotec, Auburn, CA), anti‐ANK61 antibody (Abcam, ab36392), antimouse IgG FITC (Abcam, ab97239), anti‐ANK44 (Abcamab36388), and antimouse IgG AlexaFluor 405 (Abcam, ab175663). Flow cytometry was performed on the Miltenyi MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowLogic software (Innovai, Sydney, Australia). Lymphocytes were gated in the forward and side scatter plots. After doublet exclusion, additional gates were set using fluorescence minus one (FMO) controls. Results are expressed as % of cells in the gated lymphocyte population.
Anti mouse igg fitc
Manufactured by Abcam
Sourced in Japan, United States
Anti-mouse IgG-FITC is a fluorescently labeled secondary antibody that binds to mouse immunoglobulin G (IgG). It is commonly used in immunofluorescence techniques to detect and visualize the presence of mouse primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg tr texas red, by Abcam (2 mentions)
Qcolor 3 digital camera, by Olympus (2 mentions)
Miltenyi macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Flowlogic software, by Miltenyi Biotec (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Guava easycyte ht flow cytometer, by Merck Group (1 mentions)
Cytomics fc 500 system, by Beckman Coulter (1 mentions)
7 aminoactinomycin d 7 aad, by Beckman Coulter (1 mentions)
Rabbit anti human tyrosine hydroxylase th, by Abcam (1 mentions)
Anti rabbit igg tritc, by Abcam (1 mentions)
Anti rabbit igg fitc, by Merck Group (1 mentions)
Anti mouse igg tritc, by Merck Group (1 mentions)
Rabbit anti human lmx1a, by Abcam (1 mentions)
Rabbit anti human nurr1, by Santa Cruz Biotechnology (1 mentions)
Vegfr2, by Abcam (1 mentions)
Nrp1 antibody, by Abcam (1 mentions)
Gp130, by Santa Cruz Biotechnology (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Dmr fluorescent microscope, by Leica (1 mentions)
7 protocols using anti mouse igg fitc
1
Isolation and Analysis of Placental Leukocytes
2
DENV-Infected Vero Cells Detection
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The percentage of DENV-infected Vero cells was detected by intracellular staining of viral protein using FACS. Briefly, adherent Vero cells infected with DENV for 72 h were removed from each well with trypsin-EDTA, washed with PBS once, and subsequently treated with 1× BD lysing solution (BD Biosciences) and permeabilization buffer (eBioscience) at RT for 10 min; then, anti-DENV mAb D1-11 (Abcam) at 1:500 dilution was added to each tube for 40 min at RT. Following washing once with PBS, the cells were incubated with 1:200 diluted anti-mouse IgG-FITC (Abcam ab6785) for 30 min at RT, then washed once with PBS, fixed with 4% paraformaldehyde solution, and subjected to FACS analysis (Millipore, Guava easyCyte HT flow cytometer). Data were analyzed using FlowJo software.
3
Cell Cycle Analysis by Immunofluorescence Flow Cytometry
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HEC-50B and Ishikawa cells were seeded in 60-mm culture dishes at a density of 5×105 cells/dish, collected at various time points, and fixed using ice-cold 100% methanol. Subsequently, the expression of cyclin A was analyzed using direct-immunofluorescence flow cytometry, which was performed through double-staining with a combination of fluorescein isothiocyanate (FITC)-conjugated anti-human cyclin A2 mouse antibody (clone 11B2G3) and 7-amino-actinomycin D (7-AAD); both reagents were from Beckman Coulter (Brea, CA, USA). Ki-67 analysis was performed through single staining with an anti-mouse Ki-67 monoclonal antibody (clone PP-67, 1:100) and anti-mouse IgG/FITC (1:200); both antibodies were from Abcam (Tokyo, Japan). Cell suspensions were incubated with anti-cyclin A2 and 7-AAD for 20 min at room temperature (20-25°C) and washed; suspensions were then incubated with anti-Ki-67 for 20 min at room temperature and washed. Lastly, the cells were incubated with anti-mouse IgG/FITC for 20 min at room temperature and then washed. The percentage of cells in each cell-cycle phase was determined through flow cytometry performed on a Cytomics FC 500 system (Beckman Coulter). In each experiment, an isotype-matched irrelevant mouse antibody was used as a negative control.
4
Immunofluorescence Characterization of hADSCs
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hADSCs were differentiated in 24-well chambers at 29 days, rinsed three times with PBS, and incubated in 4% paraformaldehyde overnight at 4°C. Bovine serum albumin (1%) in PBS was used for blocking. Afterwards, the cells were incubated overnight at 4°C with the following primary antibodies: mouse anti-human Cav-1 (1:100; Cell Signaling Technology), rabbit anti-human tyrosine hydroxylase (TH) (1:100; Abcam), rabbit anti-human Lmx1a (1:100; Abcam) or rabbit anti-human Nurr1 (1:100; Santa Cruz Biotechnology). The samples were then rinsed three times thoroughly with PBS and incubated with the following secondary antibodies: anti-rabbit IgG-FITC (Sigma-Aldrich), anti-mouse IgG-FITC (Abcam), anti-mouse IgG-TRITC (Sigma-Aldrich) or anti-rabbit IgG-TRITC (Abcam) for 1.5 hours at room temperature. The cells were thereafter rinsed three times in PBS and incubated for 5 minutes with Hoechst 33258. The samples were then washed twice with PBS and once in deionized water. Stained cells were observed under a confocal laser scanning microscope (SP8, Leica).
5
Immunofluorescence Assay for NRP-1 and VEGFR-2
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Primary antibodies were a rabbitmonoclonal NRP-1 antibody (1:200, Abcam, Cambridge, MA), a mouse monoclonal VEGFR-2 (1:50, Abcam, Cambridge, MA). Primary antibodies were detected by using secondary antibodies of anti-rabbit IgG-TR(Texas Red) (Abcam, Cambridge, MA), anti-mouseIgG-FITC (Abcam, Cambridge, MA), respectively. L02 were cultured on attachment factor-coated slide wells (Sonic Seal Slide Well, Nalge Nunc International). Cells were incubated with primary antibodies overnight at 4°C. Sections were washed three times in PBS, followed by secondary antibody for 1h at room temperature. Samples were analyzed with an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).
6
Immunofluorescence Analysis of gp130 and IL-6
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The primary antibodies used were rabbit polyclonal gp130 (1:100; Santa Cruz, CA, USA) and rabbit monoclonal IL-6 (1:200; Abcam, Cambridge, MA, USA) antibodies. The primary antibody was detected by the secondary antibodies of anti-rabbit IgG-TR (Texas Red; Abcam) and anti-mouse IgG-FITC (Abcam). Cells were incubated with the respective primary antibody overnight at 4 °C. Sections were washed three times in PBS, followed by incubation with the secondary antibody for 1 h at room temperature. Samples were analyzed with an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).
7
Immunohistochemical Analysis of Muscle Tissues
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Immunohistochemical analysis was performed as described in a previous study [20 (link)]. Briefly, the TA muscle was dissected, post-fixed, dehydrated, and sectioned (8-μm-thick sections) using a cryostat. The sections were thaw-mounted onto poly-l -lysine-coated slides and stored at −20 °C prior to immunostaining. The slides were washed in phosphate-buffered saline (PBS) for 10 min at room temperature, blocked, and then incubated overnight at 4 °C with primary antibodies. After washing with PBS, the slides were incubated at 4 °C for 24 h with two secondary goat antibodies labeled with fluorescein (FITC): anti-mouse IgG-FITC (1:800; Abcam, Cambridge, MA, USA) and anti-rabbit IgG-cy3 (1:1000; Abcam, Cambridge, MA, USA). The slides were washed 3 times in PBS, covered with cover slip and visualized under a DMR fluorescent microscope (Leica Microsystems, Wetzlar, Germany).
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