Transfected cells grown on glass coverslips were fixed for 30 min in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (Promega) in PBS buffered saline for 30 min. After washing with TBS-0.1% Triton X-100 (TBSTx), nonspecific binding sites were blocked with TBSTx-5% BSA (Roche) for 60 min. Then cells were applied sequentially with a 1:100 dilution of rabbit polyclonal antibody anti-OPN (Abcam) at 4°C overnight and a 1:200 dilution of Cy3-conjugated goat anti-rabbit IgG (Beyotime) at room temperature for 60 min in the dark. For negative control, PBS was added instead of primary antibody. Further, immunofluorescence was visualized under a laser-scanning confocal microscope (Zeiss, Oberkochen, Germany).
Cy3 conjugated goat anti rabbit igg
Manufactured by Beyotime
Sourced in China
Cy3-conjugated goat anti-rabbit IgG is a secondary antibody used for immunodetection applications. It is produced by conjugating Cy3 dye to goat-derived antibodies that specifically recognize and bind to rabbit immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (7 mentions)
Fitc conjugated goat anti mouse igg, by Beyotime (3 mentions)
Confocal microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Triton x 100, by Promega (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Anti opn, by Abcam (1 mentions)
Cy3 conjugated goat anti mouse igg, by Beyotime (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Beyotime (1 mentions)
Rabbit anti aqp4 antibody, by Proteintech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Evos fl auto 2 microscope, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole dapi c1002, by Beyotime (1 mentions)
Tcs sp8 x, by Leica (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Beyotime (1 mentions)
24 protocols using cy3 conjugated goat anti rabbit igg
1
Immunofluorescence Staining of OPN
2
Vimentin Role in M. pneumoniae Adhesion
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To determine whether vimentin is a key receptor protein for the adhesion of M. pneumoniae and rP1-C to host cells, a series of adhesion and adhesion inhibition experiments were performed with the following groups: (i) blank control groups, (ii) anti-vimentin antibody preincubation group, and (iii) vimentin preincubation group. The cells were added to 24-well plates (5 × 105 cells/well) and incubated at 37°C for 24 h. After cultivation for 48 h, group i was incubated with rP1-C (0.5 mg/mL) or M. pneumoniae (5 × 105 CCU/mL) for 2 h. Group ii was preincubated with mouse anti-vimentin antibody (1:200) or rabbit anti-vimentin antibody (1:200) before incubation with rP1-C or M. pneumoniae , and group iii cells were incubated with rP1-C or M. pneumoniae that had been pretreated with vimentin (0.2 mg/mL). Nonimmune rabbit or rat IgG served as a control. Anti-rP1-C antibody and anti-M. pneumoniae antibody were incubated at 37°C for 2 h. Cy3-conjugated goat anti-rabbit IgG (1:200; A0516; Beyotime) or Cy3-conjugated goat anti-mouse IgG (1:200; A0521; Beyotime) was then added to the wells, followed by incubation for 1 h at 37°C. After staining with DAPI, the cells were imaged using a laser scanning confocal microscope (LSCM; Zeiss, Germany).
3
Immunofluorescence Analysis of LC3-II
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Briefly, the cells in each group were plated onto coverslips and cultured overnight with or without LIG. Afterwards, the cells were fixed with 4% paraformaldehyde (Beyotime, Wuhan, China) for 40 minutes at room temperature and washed with phosphate-buffered saline containing 0.1% Triton X-100 (Beyotime). The cells were incubated with primary antibodies (rabbit anti-microtubule-associated protein l light chain 3B (LC3-II) polyclonal antibody; 1:400; #ab48394, Abcam, Cambridge, UK) overnight, followed by Cy3-conjugated goat anti-rabbit IgG (1:400; #A0516, Beyotime) at 37°C for 1 hour. The cells were imaged under a confocal microscope (Olympus, Tokyo, Japan) and analyzed using Image J software (NIH, Bethesda, Maryland, USA).
4
Immunohistochemical Analysis of Aquaporin-4 and GFAP Expression
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After being deparaffinized and rehydrated, the slices were antigen-retrieved in antigen retrieval buffer and blocked with goat serum. Then, the slices were incubated with AQP4 antibody (rabbit anti-AQP4 antibody; 1: 50 diluted in PBS; Proteintech) and glial fibrillary acidic protein (GFAP) antibody (mouse anti-GFAP antibody; 1: 50 diluted in PBS; Santa Cruz, Dallas, TX, USA) overnight at 4°C. After being rinsed in PBS, the slices were incubated with corresponding secondary antibodies (Cy3-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG; 1: 200 diluted in PBS; Beyotime) for 90 min at room temperature in the dark. Thereafter, the slices were rinsed in PBS and incubated with DAPI (4′,6-diamidino-2-phenylindole) (Beyotime) for nuclear counterstaining. The slices were observed using a fluorescence microscope (OLYMPUS; model: BX53) with a 400× magnification.
5
Immunofluorescence Staining of α-SMA
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Cells grown on glass slides were fixed with 4% paraformaldehyde for 20min, washed with PBS, permeabilized with 0.1% Triton X-100 at room temperature for 5min, and then blocked with 1% BSA (BSA, #A3912, Sigma) for 30min to inhibit non-specific binding. Next, the cells were incubated overnight with α-SMA antibody (#ab124964, Abcam) at 4°C. The next day, the α-SMA antibody was removed, and cells were incubated with Cy3-conjugated goat anti-rabbit IgG (#A0516, Beyotime) at room temperature for 1h. The nucleus was counter stained with DAPI (#C1002, Beyotime). Images were taken using an Evos FL Auto2 microscope (Thermo Fisher Scientific).
6
Immunofluorescence Staining of Cell Markers
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The indicated cells were seeded in 24-well plates and incubated for 24 h before being stained. For these experiments, in which we stained the cells for E-cadherin and vimentin, we fixed the cells in 4% paraformaldehyde and then permeabilized them in 0.1% Triton X-100 at room temperature. After being blocked for 1 h with blocking solution, the cells were incubated with the appropriate primary antibodies overnight at 4 °C, after which they were incubated with FITC or Cy3-conjugated goat anti-rabbit IgG (Beyotime, China) at 37 °C before being counterstained with DAPI to visualise the nuclei. Fluorescence was imaged using a Leica microscope.
7
Immunofluorescent Analysis of Cell Spheroids
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The cell spheroids were washed with phosphate-buffered saline (PBS) supplemented with 0.5% Tween20 (PBST) and fixed in methyl alcohol. Subsequently, the spheroids were blocked with 5% bovine serum albumin for 2 h at room temperature. Cells were then incubated with rabbit anti-CD133 and EpCAM antibodies (dilution 1:100) at 4°C overnight. After being washed with PBST, cells were incubated with cy3-conjugated goat antirabbit IgG (dilution 1:200) (Beyotime). 4′,6-Diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO, United States) was used to stain the nucleus for 15 min. The fluorescent images were captured using a fluorescence microscope (Olympus IX-70, Tokyo, Japan).
8
Immunofluorescence Assay for Pyroptosis
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The pyroptosis of Raw 264.7 cells was tested by immunofluorescence assay. Briefly, cells were grown on glass coverslips, washed thrice with PBS for 3 min, fixed with PBS containing 4% formaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 20 min. After washing with PBS thrice for 3 min, cells were incubated overnight at 4 ℃ with the antibody against GSDMD (Abcam, UK). They were then washed with PBS and incubated with Cy3-conjugated goat anti-rabbit Ig G (Beyotime Biotechnology, China). Nuclei were stained with DAPI (Beyotime Biotechnology, China). Finally, coverslips were observed using a fluorescence microscope.
9
Immunofluorescence Analysis of Metastatic Lung Tissues
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Metastatic lung tissues were embedded in paraffin and sectioned, and then immunofluorescence staining was carried out according to a published protocol.25 (link) Primary Abs against E‐cadherin (1:200; 20874‐1‐AP) and vimentin (1:300; 10366‐1‐AP) were obtained from Proteintech. The DAPI DNA dye was obtained from Beyotime Biotechnology. The secondary Ab was a cy3‐conjugated goat anti‐rabbit IgG (Beyotime Biotechnology). All stained tissues were examined and photographed under a NEXcope (NE900) confocal fluorescence microscope (Ningbo Yongxin Optics Co., Ltd).
10
rFgTPx Localization in Goat PBMCs
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Freshly collected goat PBMCs were treated with rFgTPx for 1 h at 37 °C in a humidified atmosphere with 5% CO2. The cells were pelleted by centrifugation at 300× g for 10 min and washed three times in PBS to remove unbound proteins. The goat PBMCs were fixed in 4% paraformaldehyde for 10 min at ambient temperature, washed three times in ice-cold PBS (5 min each), and then blocked with PBS containing 4% BSA for 1 h. The rFgTPx–treated or non–treated (control) goat PBMCs were incubated with primary rabbit anti–rFgTPx antibody (1:500) for 1 h at 37 °C. After incubation, the cells were washed three times in PBS and stained with Cy3-conjugated goat anti-rabbit IgG (1:500) (Beyotime, Haimen, Jiangsu, China). The cells were washed five times after being incubated with secondary antibody at 37 °C for 1 h. Hoechst 33,342 (Invitrogen, Eugene, OR, USA) was used to counterstain the cell nuclei. Immunofluorescence-labeled cell preparations were analyzed using a Zeiss laser scanning microscope (LSM710, Zeiss, Jena, Germany) with the 63× oil-immersion objective and Zeiss operating system associated with the ZEN imaging program.
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