We reseeded cells in cell culture dishes. Then, we fixed cells with 4% PFA and treated them with blocking solution (1% goat serum and 0.5% Triton X-100). The cells were stained with rabbit anti-human O-linked N-acetylglucosamine (RL2) (MA1-027, Thermo) over night at 4 °C. Then, the cells were washed three times with PBS and incubated with FITC-conjugated goat anti-rabbit IgG secondary antibody (A24532, Invitrogen) for 1 h at room temperature. The cell nuclei were stained with 0.1 mg/ml DAPI (Invitrogen). Finally, the stained cells were examined with a confocal microscope.
Fitc conjugated goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
The FITC-conjugated goat anti-rabbit IgG secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in various immunoassays. The antibody is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate), which allows for the visualization and quantification of rabbit IgG proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 mouse anti human cd31, by BD (1 mentions)
Bovine serum album, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Image pro plus version 6, by Media Cybernetics (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Confocal microscopy, by Leica (1 mentions)
Cy3 labeled goat anti mouse igg, by Beyotime (1 mentions)
Nf κb p65 rabbit polyclonal antibody, by Beyotime (1 mentions)
Bovine serum albumin bsa, by Bovogen (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv10i confocal microscope, by Olympus (1 mentions)
7 protocols using fitc conjugated goat anti rabbit igg secondary antibody
1
Immunofluorescence Staining for O-GlcNAc
2
Immunofluorescence Staining of MIF in Cells
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Cells were reseeded in cell-culture dishes. After induction, the cells were fixed in 4% PFA and treated with a blocking solution containing 1% goat serum and 0.5% Triton X-100. The cells were stained with rabbit anti-human MIF antibody (Abcam, ab175189) overnight at 4°C. The cells were then washed three times with PBS and incubated with FITC-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, A24532) for 1 hr at room temperature. Subsequently, cell nuclei were stained with 0.1 mg/mL DAPI (Invitrogen). The stained cells were examined with a confocal microscope.
3
Whole-mount immunofluorescence staining of cell-loaded scaffolds
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Whole-mount IF staining of the cell-loaded scaffolds was performed as described previously (Wang et al., 2010 (link)). In brief, the samples were collected at the indicated time points and fixed in 4% paraformaldehyde for 12 h. After thoroughly washing with PBS solution, the scaffolds underwent sequential membrane permeabilization and blocking with nonspecific antigens. Subsequently, they were incubated overnight at 4°C using the following primary antibodies: mouse anti-human ALB (dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-human CYP3A4 (dilution, 1:100; Proteintech, China), rabbit anti-human MRP2 (dilution, 1:50; Proteintech), and Alexa Fluor®647 Mouse anti-Human CD31 (dilution, 1:100; BD Biosciences). Samples not incubated with specific primary antibodies served as negative controls. Next, cell-loaded scaffolds were incubated with FITC-conjugated Goat anti-Rabbit IgG Secondary Antibody or FITC-conjugated Donkey anti-Mouse Secondary Antibody (dilution, 1:200; Invitrogen) for 2 h at room temperature. DAPI (2 μg/ml, Research Organics, Cleveland, OH, United States) was used to counterstain the cell nucleus, and images were captured using CLSM.
4
Immunophenotyping of Isolated Cells
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In order to characterize the isolated cells, S100β, P75NTR and GFAP immunostaining was performed. The cells were fixed to slides with 4% paraformaldehyde (PFA) for 15 min at room temperature, washed three times with PBS, treated with 0.3% Triton X-100 to permeabilize the membranes, washed again in PBS and blocked with 10% bovine serum album (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. The slides were subsequently incubated with rabbit anti-S100 polyclonal antibody (1:200), rabbit anti-P75NTR polyclonal antibody (1:500) or rabbit anti-GFAP polyclonal antibody (1:500) at 37°C for 1 h. The slides were washed three times with PBS and incubated with a FITC-conjugated goat anti-rabbit IgG secondary antibody (1:500; cat no. F2765) for 1 h at 37°C, and finally incubated with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 min at room temperature to stain the cell nuclei. Images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and processed using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Cells in three randomly selected fields were counted to calculate the SC purity.
5
Immunofluorescence Analysis of NF-κB Activation
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Mv.1.Lu cells were cultivated on cover glasses in 24-well plates, followed by infection with PS or CDV3 at an MOI of 2 when the cells reached ~70% confluence. The mock-infected cells were treated with PBS as a negative control. Next, at 24 hpi, the cells were fixed with 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Further, the cells were incubated with an NF-κB P65 rabbit polyclonal antibody (Beyotime, China) and a mouse monoclonal antibody specific to CDV N protein and incubated with Cy3-labeled goat anti mouse IgG (Beyotime, China) and FITC-conjugated goat anti-rabbit IgG secondary antibody (ThermoFisher, U.S.A) prior to staining with DAPI. The fluorescent images were analyzed under confocal microscopy (Leica, Germany).
6
Immunolocalization of Estrogen Receptors in Mouse Embryos
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Embryos at zygote stage or 2-cell/4-cell/8-cell/morula satge were collected from the oviducts following different timing (20 h, 48 h, 54 h, 65 h and 72 h respectively) after HCG injection. Blastocysts were collected from the uterine cavity following 96 h after HCG injection. Embryos collected at different stages were fixed immediately by incubating in 4% paraformaldehyde for 30 min at room temperature. Samples were permeabilized by incubating with 0.5% Triton X-100 and 1% BSA in PBS, and then were washed three times with PBS containing 0.2% Triton X-100 and 0.3% BSA. Therefore, samples were incubated in blocking buffer containing 4% BSA and 0.2% Triton X-100 in PBS at room temperature for 1 h, followed by incubation overnight at 4 °C with rabbit anti-mouse ERα (PA1-309) and ERβ (PA1-311) primary antibodies (Thermo Scientific, Waltham, MA, USA), diluted 1:100. Samples were then incubated at room temperature for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody (31635; Thermo Scientific), diluted 1:100, following by counterstaining with bisbenzimide (BIS) to label nuclei.
7
Immunofluorescence Staining Protocol
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Immunofluorescence staining was performed as described previously [56 (link)] with some modifications. Cells were fixed with 1% paraformaldehyde (PFA) (Sigma-Aldrich) solution at room temperature for 15 min and permeabilized with 0.1% Triton X-100 (Merck, Darmstadt, Germany) for 10 min. After several washes with 1 × PBS, the fixed cells were blocked using 3% bovine serum albumin (BSA; Bovogen Biologicals, VIC, Australia) and 5% FBS, and subsequently incubated with indicated monoclonal antibodies (1:100) at 4 °C overnight. Cells were washed thrice with PBS and incubated with the cyanine 3 (Cy3)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. Samples were counterstained with 100 μL DAPI. Finally, cells were mounted and observed using a fluorescent or FV10i confocal microscope (Olympus, Center Valley, PA, USA).
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