After treatment, cells were fixed with 4% paraformadehyde and permeabilized using 0.2% Triton X-100. After blocking the non-specific binding with 5% FBS in PBS, cells were incubated with 1:100 anti-NF-κB (Santacruz) at 4 °C, overnight. The reactivity was detected by incubation with 1:100 anti-rabbit-IgG-FITC (Santa Cruz) for 1 h at room temperature. Nuclei were stained with 1:10,000 Hoechst 33342 (Molecular probe, invitrogen, UK) and the fluorescence signal was observed under the Nikon fluorescence microscope (Nikon Corporation, Tokyo, Japan).
Anti rabbit igg fitc
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-rabbit IgG-FITC is a fluorescently labeled secondary antibody that binds to rabbit primary antibodies. It can be used in various immunodetection techniques, such as Western blotting, ELISA, and immunofluorescence microscopy, to visualize the presence and distribution of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Sc 2012, by Santa Cruz Biotechnology (2 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Anti nf κb, by Santa Cruz Biotechnology (1 mentions)
Anti gamma h2a x phospho s139 antibody, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Vector Laboratories (1 mentions)
Anti mouse igg fitc, by Bio-Rad (1 mentions)
Ix81 fluorescence microscope, by Olympus (1 mentions)
Laser confocal microscope, by Olympus (1 mentions)
Diaminophenylindole, by Merck Group (1 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
Mouse anti claudin 1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Bz x710, by Keyence (1 mentions)
Vectashield mounting medium with dapi h 1200, by Vector Laboratories (1 mentions)
Ab104139, by Abcam (1 mentions)
12 protocols using anti rabbit igg fitc
1
Immunofluorescence detection of NF-κB
2
Quantifying DNA Damage in HaCaT Cells
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HaCaT cells were seeded in 6-well plates. Six hours or 24 h after SM exposure and ABT-888 administration, the cells were harvested, permeabilized and fixed. The primary antibody was the anti-gamma H2A.X (phospho S139) antibody (Abcam) and the secondary antibody was anti-rabbit IgG-FITC (Santa Cruz Biotechnology). A FACSCalibur flow cytometer (Becton, Dickinson and Company, BD) was used to detect the fluorescence intensity.
3
Immunohistochemical Analysis of Gremlin Expression in Pituitary Adenomas
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Pituitary adenoma tissues samples were selected from 60 subjects, including 28 women (17–76 years old) and 32 men (22–75 years old) with ACTHoma (n = 5), GHoma (n = 23), NFoma (n = 22), PRLoma (n = 6), and TSHoma (n = 4). Samples were paraffin embedded and used to build tissue microarrays that were analyzed immunohistochemically using a protocol available online (http://genome-www.stanford.edu/TMA/ ). Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β-actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA). Expression was examined by fluorescence microscopy (FMV 1000; Olympus, Tokyo, Japan). The fluorescence intensity of the negative control was subtracted and the level of Gremlin relative to β-actin was calculated as a percentage using image analysis software (Image Pro-Plus, ver. 6.3; Media Cybernetics Division of Nippon Roper, Tokyo, Japan).
4
Porcine MoDCs Immunofluorescence Analysis
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Immunofluorescence analysis of porcine MoDCs was conducted as described in our previous reports [15 (link)]. Mounted MoDCs were incubated for 20 min with 1 % bovine serum albumin and 2 % normal goat serum (Vector Laboratories, Burlingame, CA) in PBS at room temperature to block nonspecific binding sites. After removal of the blocking solution, samples were incubated for 16 h at 4 °C in a humidified chamber with one of the following primary antibodies: anti-porcine CD172a-fluorescein isothiocyanate (FITC) SWC3 IgG1 (Southern Biotech), anti-porcine CD11R1-unlabeled mouse IgG1 (AbD Serotec, Kidlington, Oxford, United Kingdom), anti-porcine major histocompatibility complex class II (MHC-II)-unlabeled mouse IgG2a, anti-porcine TLR2-unlabeled rabbit IgG (Santa Cruz); or anti-porcine TLR4-unlabeled rabbit IgG (Santa Cruz). Samples were washed in PBS and then treated with one of the following secondary antibodies: anti-mouse IgG2a-FITC (AbD Serotec), anti-mouse IgG-FITC (AbD Serotec), or anti-rabbit IgG-FITC (Santa Cruz), when necessary.
5
Quantifying pERK1/2 Expression in HRMECs
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To determine the expression levels of pERK1/2 in HRMECs, Gas6 treated HRMECs were fixed in 2% paraformaldehyde. Cells were permeabilized and blocked with 0.1% Triton X-100/PBS containing 2% BSA and 0.5% normal serum to reduce the nonspecific adherence of antibodies. Cells were incubated in primary anti-pERK1/2 at a dilution of 1∶1000 for 1 h at 37°C in a humidified chamber. After incubation with primary antibody, the cells were rinsed and incubated with anti-rabbit IgG -FITC (Santa Cruz Biotechnology). Cell nuclei were stained with Hoechst stain (blue). After washing with PBS and treating with mounting solution (Wako, Kumamoto, Japan), cells were observed under an Olympus IX-81 fluorescence microscope using a 20× objective.
6
Immunofluorescent Visualization of CCNG1
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5-μm frozen section from each sample was fixed in acetone at 4 °C overnight. After washing, sections were blocked with 1 % bovine serum albumin for 30 min and incubated overnight at 4 °C with rabbit anti-human CCNG1 antibody (1:50). Then sections were incubated with anti-rabbit IgG–FITC (1:100, Santa Cruz Biotechnology) for 2 h at room temperature in the dark. Nuclei were stained with diaminophenylindole (1 μg/mL; Sigma-Aldrich) for 5 min at room temperature. Coverslips were mounted and imaged using a laser confocal microscope (Olympus, Tokyo, Japan).
7
Immunofluorescence Imaging of Colon Tissue
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The 5 µm-thick frozen colon tissue and cells on the slides were fixed in an acetone-methanol (1:1) mixture at -20℃. The slides were incubated with primary antibodies (anti-rabbit ZO-1, anti-mouse Occludin, anti-mouse Claudin-1; Invitrogen, Waltham, MA) overnight at 4℃ and secondary antibodies (anti-rabbit IgG FITC, Santa Cruz; Alexa Fluor 488 goat anti-mouse IgG; Invitrogen) for 2 hours at room temperature. After mounting the slides with VectaShield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA), the slides were sealed with nail polish and stored at -20℃ until observation, using a confocal microscope (Nikon Instruments Inc., Melville, NY).
8
Immunofluorescence Analysis of Skin Proteins
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The skin samples were fixed with 10% buffered formalin overnight. After paraffin embedding, 3-μm sections were deparaffinized and subjected to antigen retrieval and blocking with PBS (pH 7.2) containing 10% goat serum, followed by 3 hours of incubation at room temperature with diluted primary antibodies (ABCA12, 2 μg/ml, bs-11906R; Bioss, Woburn, MA; CDSN, 4.7 μg/ml, 27953-1-AP; Proteintech, Rosemont, IL). Antibody binding was visualized by incubation with a secondary antibody (anti-rabbit IgG-FITC, 2 μg/ml, sc-2012; Santa Cruz Biotechnology, Santa Cruz, CA) for 60 minutes at room temperature, followed by mounting with medium containing DAPI (VECTASHIELD mounting medium with DAPI, H-1200; Vector Laboratories, Burlingame, CA). The samples were examined using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).
9
Immunofluorescence Staining of SH-SY5Y Cells
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SH-SY5Y expressing ROP16 were seeded onto 13-mm circular glass coverslips with regard to the immunofluorescence studies,. The next day, cells were washed with PBS, warmed to 37°C, and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were washed three times with PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 mins. Nonspecific staining was reduced by blocking with 5% goat serum overnight at 4°C and then followed by incubation with anti-rabbit IgG-FITC and anti-mouse IgG-Cy3 (Santa Cruz) as the secondary antibodies.
10
Immunofluorescence analysis of CBR1 in OGD-treated HepaRG cells
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HepaRG cells were seeded onto poly-L-lysine coated glass (GG-18-PLL; Neuvitro, USA) and treated OGD for 7 h followed by 2 h of recovery. After washing with ice-cold PBS 3 times, cells were fixed with 4% formaldehyde. Cells were permeabilized in PBS with 0.2% NP-40, 1% bovine serum albumin (BSA), and 0.1% sodium azide and washed 3 times with ice-cold PBS. The primary antibody, anti-CBR1 (1:100, ab156590; Abcam, UK), was diluted into PBS with 1% BSA and 0.1% NP-40 for 10 min, followed by incubation with the secondary antibody, anti-rabbit IgG-FITC (1:100, sc-2012; Santa Cruz Biotechnology, USA). After washing with PBS 3 times, cells were mounted using DAPI mounting solution (ab104139; Abcam). All images were observed under a fluoroscopic microscope (AxioObserver Z1; Carl Zeiss, Germany).
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