The replications of H1N1 virus in the cells were determined after detecting expression of IAV NP by immunofluorescence staining. Cultured cells were twice rinsed with PBS (0.01 M, pH 7.2) and then were fixed with 4% paraformaldehyde solution for 15 min at room temperature. After rinsing with PBS, the cells were permeabilized with 0.5% Triton X-100 for 15 min, and then rinsed and blocked with 3% BSA for 20 min at room temperature. The cells were incubated with mouse anti-IAV NP monoclonal antibody (1:1000, Abcam, Shanghai, China) overnight at 4°C. After rinsing three times with PBS, the cells were incubated with a FITC-conjugated goat anti-mouse secondary antibody (1:500, Abcam, Shanghai, China) for 1h at room temperature. To visualize the nuclei, cells were stained with 3 μg/ml 40, 60—diamidine-2-phenylindole (DAPI) (Sigma-Aldrich, Shanghai, China) for 5 min at room temperature and then they were examined under an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
Fitc conjugated goat anti mouse secondary antibody
Manufactured by Abcam
Sourced in China, United States, United Kingdom, Japan
FITC-conjugated goat anti-mouse secondary antibody is a laboratory reagent used for detection and visualization purposes. It is a secondary antibody that binds to mouse primary antibodies and is conjugated to the fluorescent dye fluorescein isothiocyanate (FITC).
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Summit software, by Agilent Technologies (1 mentions)
Cyan adp flow cytometer, by Agilent Technologies (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Ts100, by Nikon (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
6 protocols using fitc conjugated goat anti mouse secondary antibody
1
Quantifying IAV NP Expression in Cells
2
Immunofluorescence Staining of ALOX15 and CD68
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Cells were fixed with 4% paraformaldehyde for 10 min, permeabilization with 0.2% Triton-X 100 for 10 min at 4 °C, and blocked with 3% bovine serum albumin for 1 h at room temperature, followed by incubation overnight at 4 °C with ALOX15 (Abcam ab119774, 1:100) and CD68 (CST, #23308, 1:150). Cells were washed 3 times for 5 min in PBS, and then incubated for 2 h with FITC-conjugated goat anti-mouse secondary antibody (diluted 1:200 in blocking buffer; Abcam). Acquired images with a laser scanning confocal microscope.
3
Bovine Immune Marker Evaluation
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To evaluate immune markers, the cells that had been seeded in 96 well round-bottom plates were centrifuged, supernatant was discarded, and cells were resuspended in PBS with 5% Bovine Serum Albumin, which also served as blocking solution for flow cytometry. The cells were then incubated for 30 min with primary anti-bovine antibodies (diluted 1:200) for anti-bovine MHC I (VMRD, USA) or MHC II (VMRD), washed with PBS three times, incubated for 30 min with a FITC-conjugated goat anti-mouse secondary antibody (AbCam, Cambridge, MA, USA) (diluted 1:200), fixed with 2% paraformaldehyde, and then analyzed by flow cytometry. Control samples from each fetus were incubated in blocking solution without primary antibodies. Data were collected for 10,000 events on a Cyan-ADP Flow cytometer (Dako, Ft. Collins, CO, USA) and analyzed with Summit software (Dako, Ft. Collins, CO, USA).
4
PTRF and TLR4 Immunofluorescence Imaging
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Cells were cultured in confocal dishes and washed with PBS three times, followed by fixation in 4% paraformaldehyde for 30 min, permeabilization with 0.5% Triton X-100, and blocking with goat serum. Then, the cells were subjected to staining with the anti-PTRF antibody (Thermo Invitrogen) at a dilution of 1:50, followed by the CY3-conjugated goat anti-rabbit secondary antibody (Abcam) at a dilution of 1:500 prior to imaging. For paraffin slices obtained from colon tissue samples, the PTRF antibody (1:50, Thermo Invitrogen) and TLR4 antibody (1:50, Thermo Invitrogen) were used as primary antibodies, followed by the FITC-conjugated goat anti-mouse secondary antibody (1:500, Abcam) or the CY3-conjugated goat anti-rabbit secondary antibody (1:500, Abcam) for imaging. The cells and slides were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) as a nuclear indicator and visualized with a confocal laser-scanning microscope (LSM 880 Carl Zeiss).
5
Collagen I and II Immunohistochemistry
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Specimens were mounted by use of O.C.T. compound (Tissue-Tek, Miles, USA). They were cut into cryosections 10 μm thick and stained with Safranin O and collagen I and II immunohistochemistry for observation of cell proliferation and substrate secretion in vivo. The process of collagen I and II immunohistochemistry was as follows: the cryosections were washed with phosphate-buffered saline (PBS), inactivated for endogenous peroxidase with 3% hydrogen peroxide in PBS for 15 min at room temperature, and blocked with 1% bovine serum albumin in PBS for 30 min to prevent nonspecific binding of proteins. Subsequently, the cryosections were incubated with monoclonal anti-collagen I or II antibody (Abcam, UK) diluted 1:100 in PBS at 4°C overnight in a humidified chamber, and then stained with goat anti-mouse FITC-conjugated secondary antibody (Abcam, UK) for 2 h at room temperature. Slides were then treated with 3,3'-diaminobenzidine (DAB, Sigma, USA) for approximately 10–15 min until color developed. Stained cryosections were observed by inverted microscope (TS100, Nikon, Japan).
6
Immunofluorescence Assay for Bladder Cancer
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Bladder cancer cells were seeded into 48-well plates at 6000 cells/well and treated as described for the cell viability assays. After treatment for the indicated times, the cells were fixed with 4% formaldehyde for 15 min, washed with PBS, treated with 5% BSA for 30 min at room temperature, and incubated with mouse anti-human vimentin or anti-human E-cadherin primary antibodies (Cell Signaling Technology) at 4°C overnight. The cells were incubated with goat anti-mouse FITC-conjugated secondary antibody (Abcam) at 4°C for 2 h, incubated with DAPI (Sigma-Aldrich) for 2 min at room temperature, washed twice with PBS, and observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).
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