Blood samples (25 μL) by retro-orbital sinus bleeding were obtained on a weekly basis, five days after each immunization. IgG sensitization autoantibodies levels on the RBCs were determined by flow cytometry using FITC–conjugated anti–mouse IgG (Invitrogen, Life Technologies, Grand Island, NY). For analysis of rat RBC-specific xenoantibodies, rat RBCs were incubated with diluted mouse plasma for one hour at 37 °C and after several washes, were stained with FITC-conjugated anti–mouse IgG as previously described39 (link).
Fitc conjugated anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC-conjugated anti-mouse IgG is a laboratory reagent that detects and binds to mouse immunoglobulin G (IgG) antibodies. The FITC (fluorescein isothiocyanate) conjugate allows for the fluorescent labeling of the target mouse IgG molecules, enabling their visualization and detection in various applications.
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Lab products found in correlation
Alexa fluor 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Facscalibur, by BD (2 mentions)
Igg free bsa, by Jackson ImmunoResearch (1 mentions)
Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscanto, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Aipathwell softwell, by Wuhan Servicebio Technology (1 mentions)
Sj1909 nbp2 67416, by Novus Biologicals (1 mentions)
Af7244, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Alexa 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fv10i, by Olympus (1 mentions)
Annexin 5 conjugated alexa fluor 488 apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated anti mouse and anti rabbit igg, by GE Healthcare (1 mentions)
Trichostatin a tsa, by Cayman Chemical (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Fitc conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
23 protocols using fitc conjugated anti mouse igg
1
Quantitative Analysis of Xenoantibodies
2
Detecting Antibodies Against Melanoma Cells
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Indirect ELISA was used to detect antibodies against CCH in sera of treated mice.28 (link) Antibodies against melanoma cells or peripheral blood leucocytes (PBLs) were analyzed by flow cytometry. In brief, 1×105 B16F10 or Mel1, Mel2 and Mel3 cell mixtures were incubated with 1:20 dilutions of de-complemented sera. Cells were then incubated with FITC-conjugated anti-mouse IgG (Invitrogen). The binding of serum IgG to target cells was evaluated by flow cytometry.
3
Immunofluorescence Detection of HuR and P-bodies
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Cells grown on coverslips were washed twice with PBS containing 10 mM glycine, fixed in 2% paraformaldehyde for 15 min at RT, and permeabilized with 0.02% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 5% normal goat serum in PBS containing 1% IgG-free BSA (Jackson Immunoresearch) and incubated with primary antibody overnight at 4°C diluted in blocking solution. HuR was detected with anti-HuR monoclonal primary antibody (3A2, 1:200 dilution; Santa Cruz Biotechnology) for 1 hr at RT. Secondary antibody incubation was done for 1 hr at RT using FITC-conjugated anti-mouse IgG (1:100 dilution) or Alexa488-conjugated anti-mouse IgG (1:500 dilution; Invitrogen). Cells were counter-stained with DAPI to visualize nuclei. Fluorescence microscopy and image analysis was accomplished as described [31 (link), 61 (link)]. Detection of P-bodies and assessment of P-body co-localization with MS2-YFP was accomplished as described [31 (link)].
4
Flow Cytometric Quantification of Antibody Binding
5
Intracellular TTS Quantification in T-cells
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Tumors were excised, disrupted, and the cells were harvested and washed twice with PBS containing 2% FBS and 0.1% sodium azide to detect intracellular levels of TTS in tumor-infiltrated CD4+ and CD8+ T-cells. We used the BD Cytofix/Cytoperm Kit (BD Pharmingen) for intracellular TTS staining, following the manufacturer’s instructions. The intracellular TTS protein was detected with a mouse anti-TTS mAb (Santa Cruz Biotechnology) and FITC conjugated-anti-mouse IgG (Invitrogen). The cells were stained with Cy5 anti-mouse CD4 and PE-conjugated anti-CD8 for the surface molecule analysis. The stained cells were analyzed by flow cytometry.
6
Immunofluorescence Staining of Pulmonary Tissues
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Paraffin sections (4 μm) of pulmonary tissues were rehydrated and microwaved in citric acid buffer to retrieve antigens. After incubation with 10% BSA for 1 h, the sections were incubated with primary antibodies against integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), fibronectin and collagen-I (72026S, CST, USA) at a dilution of 1:100 at 4 °C overnight. After washes, sections were incubated with Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) for integrin β3, PKM2 and collagen-I, fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Invitrogen) for fibronectin at a dilution of 1:400, at 37 ℃, for 1 h in the dark. Finally, nuclei were counterstained with 4'6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA). Aipathwell softwell (Servicebio, China) was used to analyze the integrated optical density of the single or double stained fluorescent proteins for the semi-quantitative analysis.
7
Immunofluorescence Analysis of FEF3 Cells
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FEF3 cells (5 × 103/well) were seeded in 4-well chambered slides. After 24 h, the supernatants were replaced with normal medium or CM from tumor cells (CM/TE4 or CM/OE19), and the cells were cultured for 2 days. After stimulation, the cells were fixed with 4% paraformaldehyde in PBS for 15 min and blocked with 3% bovine serum albumin for 30 min (for FAP staining) or cold 100% methanol for 30 min on ice (for αSMA staining). The slides were incubated with a primary antibody for an hour (FAP; R&D, MAB3715) or overnight (αSMA; Abcam) on ice. After washing twice with PBS, the slides were incubated with the appropriate secondary antibody, FITC-conjugated anti-mouse IgG or Alexa 568-conjugated goat anti-mouse IgG (Invitrogen), for an hour on ice. The slides were further stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted by using Fluorescent Mounting Medium (Dako Cytomation). Then, the cells were analyzed with a confocal laser microscope (FV10i, Olympus, Tokyo, Japan).
8
Apoptosis and Protein Expression Assays
9
Immunohistochemical Analysis of Gliosis and Myelination
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Immunohistochemical analysis of gliosis [neuronalspecific glial fibrillary acidic protein (GFAP), rabbit polyclonal, 1:1,000; Dako, Glostrup, Denmark], myelination [myelin basic protein (MBP), 1:500; Abcam, Cambridge, UK], and reactive microglia (ED-1, 1:200; Abcam, Cambridge, MA, USA) was performed on deparaffinized 4-μm-thick brain sections, as described in our previous reports (1) . Briefly, brain slides were incubated with the diluted antibodies overnight at 4°C. Slides were washed three times with wash buffer (PBS/0.1% Triton X-100; Sigma-Aldrich). The brain slides were incubated with the diluted FITC-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Then slides were washed three times with wash buffer (PBS/0.1% Triton X-100). Nuclei were stained with DAPI for counterstain.
10
Antibody Characterization for Biological Research
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(i) First antibodies. The following first antibodies were used: Mouse anti-phospho-H2AX (Ser139) (#05-636), mouse anti-β-actin (#A5441) from MilliporeSigma rabbit anti-phospho- RPA32 (Thr21) (#AP1040), rabbit anti-phospho-DNA-PKcs (Ser2056) (#AP0621) and, rabbit anti-Pol κ (#A6122) from Abclonal, rabbit anti-phospho-ATR (Thr1989) (#GTX128145) and rabbit anti-polymerase η (#GTX109938) from GeneTex, mouse anti-AAV2 Rep protein (#03-61069; #03-61073) and rabbit anti-AAV2 capsid proteins (#03-61084) from ARP. Rat anti-MAAP was generated by immunizing rats with purified GST-MAAP, following a previously published protocol (86 (link)). Rabbit anti-AAP antisera were made by immunization of 2 rabbits with a peptide antigen, Cys-RSTSSRTSSARRIKDASRR (87 (link)), at Biomatik Co.
(ii) Secondary antibodies. The following secondary antibodies were used: DyLight 800 conjugated anti-rabbit IgG (#5151S) and DyLight 800 conjugated anti-mouse IgG (#5257S) (Cell Signaling, DyLight 800 conjugated anti-rat IgG (#SA5-10024) ThermoFisher, FITC conjugated anti-mouse IgG (#115-095-003), FITC conjugated anti-rabbit IgG (#111-095-003), Alexa Fluor 488 conjugated goat anti-mouse IgG (#115-545-062), and Alexa Fluor 594 conjugated goat anti-mouse IgG (#115-585-146) (Jackson ImmunoResearch).
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