The expression of cell-surface antigens was quantified by immunostaining as described previously [7 (link)]. We used the following monoclonal antibodies (anti-human): PE-conjugated CD133 (Miltenyi Biotec, Bergisch-Gladbach, Germany), PerCP-conjugated CD34 (BD Biosciences, Heidelberg, Germany), and either FITC-conjugated CXCR-4 or APC-conjugated c-Kit, APC-conjugated CXCR-2 (all R&D Systems, Wiesbaden-Nordenstadt, Germany), PE-conjugated PSGL1 (BD Biosciences, Heidelberg, Germany) or the indirect rabbit anti-human polyclonal RAGE antibody (Biozol, Eching, Germany), for which a FITC-conjugated anti-rabbit IgG antibody (Invitrogen, Karlsruhe, Germany) was used. We used a FACSCalibur flow cytometer (BD Biosciences) for flow cytometry. FACS-data analysis was performed with WinMDI 2.8 software (Scripps Research Institute, La Jolla, CA). EPC counts are expressed as percentage referred to total PMBC in each study subject.
Fitc conjugated anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The FITC-conjugated anti-rabbit IgG antibody is a secondary antibody that binds to rabbit immunoglobulin G (IgG) antibodies. The fluorescein isothiocyanate (FITC) dye is conjugated to the antibody, allowing for detection and visualization of the target rabbit IgG in various applications such as immunohistochemistry, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated cd133, by Miltenyi Biotec (1 mentions)
Percp conjugated cd34, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Rabbit anti epha2, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Tcs sp2, by Leica (1 mentions)
4 protocols using fitc conjugated anti rabbit igg antibody
1
Quantifying Cell-Surface Antigens by Flow Cytometry
2
Immunofluorescence Imaging of Transcription Factor
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Cells were grown on glass coverslips in 12-well plates. After cells were treated with TGFβ1, SCE, SolB, and/or SchB for 1 h, the cells were fixed with 3.7% formaldehyde in PBS for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 5% normal goat serum in PBS for 30 min. The cells were labeled with anti-p65/RelA antibody (Santa Cruz, CA) for overnight at 4°C and then probed with FITC-conjugated anti-rabbit IgG antibody (Invitrogen) and DAPI (Roche) for additional 1 h at RT. The cells were photographed using the FluoView 1000 confocal microscope (Olympus, Tokyo, Japan).
3
Immunofluorescence Analysis of EphA2 in HCC
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We have set up a database with specimens for HCC patients in the previous study (Jue et al., 2017a (link)). In this study we chose 10 samples confirmed with VM and without VM. VM was detected as above description. Expression of EphA2 in samples was detected as the previous report (Cho et al., 2020 (link)). For the immunofluorescence analysis, paraffin-embedded human HCC sections were stained with rabbit anti-EphA2 (Invitrogen) antibody, followed by FITC-conjugated anti-rabbit IgG antibody (Invitrogen). Positive cells were detected by confocal laser scanning microscopy (Nikon Eclipse TE2000-U). This study was approved by the institutional ethic committee of the Second People’s Hospital of Taizhou (NO. TZEYLL20180301, Taizhou, Jiangsu, China).
4
Investigating IGF-1 and Insulin Signaling in T Cells
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Cell activation was done as mentioned above. 13 mm coverslips (Mariefeld, Germany) were coated with poly-L-lysine (0.01% solution, Sigma, cat.# P4707) at room temperature. After 10 minutes, poly-L-lysine solution was removed and the cells were added and allowed to settle for 30–60 minutes at room temperature. Subsequently, 0.001 µM IGF-1 or 1 µM insulin in the presence or absence of 20 µM LY294002 were added for 15 and 30 minutes and then cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100. Then T cells were blocked with PBS containing 4% fetal bovine serum (FBS) (Biochrom) and incubated with FoxO1 (1:50), p-FoxO1 (1:50), or p-Akt (1:25) antibody for 1 hour. After that, cells were incubated with FITC-conjugated anti-rabbit IgG antibody (1:1000, Invitrogen, USA). To stain nuclei, 4, 6-diamidino-2-phenylindole-dihydro-chloride (DAPI, 1:1000, Invitrogen) was used. T cells were analyzed with confocal fluorescence microscopy (Leica TCS SP2, Germany) and images were captured and analyzed with Metaview software.
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