The immunofluorescence assay was performed as described previously [24 (link)]. Briefly, cells were fixed with acetone/methanol (1:1) for 30 min at 4 °C. After removal of the fixation solution, cells were washed once with PBS and blocked with PBS containing 1% FCS for 1 h at 37 °C. The blocking solution was discarded and an HEV capsid protein-specific rabbit hyperimmune serum (kindly provided from Rainer Ulrich, Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany; 1:500 dilution in PBS containing 1% FCS) was added. After 1 h incubation at 37 °C, the antibody was removed and cells were washed three times with PBS. FITC-conjugated anti-rabbit IgG (Sigma, Deisenhofen, Germany; 1:1000 in PBS containing 1% FCS) was added and the cells were incubated for 1 h at 37 °C. The secondary antibody was discarded and the cells were washed twice with PBS and once with distilled water. The cells were mounted with Roti®-Mount Fluor Care DAPI (Carl Roth, Karlsruhe, Germany) and analyzed using an Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany).
Fitc conjugated anti rabbit igg
Manufactured by Merck Group
Sourced in United States, Germany
FITC-conjugated anti-rabbit IgG is a laboratory reagent used to detect and visualize rabbit immunoglobulin G (IgG) in various experimental techniques. The product consists of fluorescein isothiocyanate (FITC) molecules covalently attached to anti-rabbit IgG antibodies, allowing for the specific labeling and identification of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Cy3 conjugated anti mouse igg, by Merck Group (3 mentions)
Roti mount fluorcare dapi, by Carl Roth (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Model ix71, by Olympus (2 mentions)
Planapo n 60 1.42 na dic objective, by Hamamatsu Photonics (2 mentions)
Orca er, by Hamamatsu Photonics (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Confocal microscope, by Olympus (2 mentions)
Facscalibur, by BD (2 mentions)
Normal horse serum, by Vector Laboratories (2 mentions)
Mitotracker red, by Thermo Fisher Scientific (2 mentions)
Rhodamine conjugated anti rabbit igg, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Rabbit anti smad2 3, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse actin, by Merck Group (1 mentions)
Rabbit anti phospho smad3, by Cell Signaling Technology (1 mentions)
Rabbit anti smad1 5 8, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti mouse phospho stat1, by Cell Signaling Technology (1 mentions)
27 protocols using fitc conjugated anti rabbit igg
1
Immunofluorescence Assay for HEV Capsid Protein
2
Immunofluorescence Assay for HEV Detection
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PLC/PRF/5 cells were seeded in 96 well plates and inoculated 2 weeks later with 100 µL of a 10-fold dilution series of supernatants from 6 weeks (49 d) p.i. of the second passage according to the infection protocol as described above. At 2 weeks after infection, the medium was removed and cells were fixed using acetone/methanol (1:1) for 30 min at 4 °C. Fixed cells were washed with PBS and blocked with PBS containing 1% FCS for 1 h at 37 °C. The solution was removed and a 1:500 dilution of an HEV capsid protein-specific rabbit hyperimmune serum [23 (link)] in PBS containing 1% FCS was added. After 1 h incubation at 37 °C, the antibody was removed and cells were washed three times with PBS. Thereafter, a 1:1000 dilution of FITC-conjugated anti-rabbit IgG (Sigma, Deisenhofen, Germany) in PBS containing 1% FCS was added and the cells were incubated for 1 h at 37 °C. The secondary antibody was removed and the cells were washed twice with PBS and once with distilled water. The cells were mounted with Roti®-Mount FluorCare DAPI (Carl Roth, Karlsruhe, Germany) and analyzed using an Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany).
3
Indirect Immunofluorescence Assay for SARS-CoV-2 Antigens
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The presence of SARS-CoV-2 antigens in A549 infected cells was evaluated by indirect immunofluorescence using polyclonal antibodies directed against N and S proteins of SARS-CoV-2 (Sino Biological, Beijing, China). For the immunofluorescence assay, the cells grown in an 8-well chamber slide were rinsed twice with PBS, fixed in methanol solution at room temperature for 30 min, washed three times with PBS, and then incubated with the specific anti-SARS-CoV-2 antibodies (diluted 1:60 in PBS) for 1 h at 37°C. After three washes with PBS, the secondary antibody consisted of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (diluted 1:1000 in PBS; Sigma, St. Louis, MI, USA) was added for 1 h at 37°C. After three final washings, drying, and mounting, the slides were observed under a fluorescence microscope.
4
Multiparametric Analysis of Signaling Pathways
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Rabbit anti phospho-Smad2 (Millipore, Billerica, MA, USA), rabbit anti Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti phospho-Smad3 (Cell Signaling Technology), goat anti phospho-Smad1/5/8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti Smad1/5/8 (Santa Cruz Biotechnology), rabbit anti-mouse phospho-Stat1 (Cell Signaling Technology), rabbit anti-mouse ApoA-I (Santa Cruz Biotechnology), rabbit anti-mouse Actin (Sigma, Steinheim, Germany) and HRP conjugate donkey anti-rabbit IgG (Southern Biotech, Birmingham, AL, USA) were used for western blot analysis. Rabbit anti SRB1 monoclonal antibody (Novus Biologicals, CO, USA) and FITC conjugated anti-rabbit IgG (Sigma, Steinheim, Germany) were used for flow cytometry analysis. Fc block and fluorochrome conjugated antibodies against mouse CD45, CD3, CD8, MHC-II, CD11c and TNF-alpha were purchased from BD Biosciences. Intracellular staining for TNF-alpha was performed following manufacturer's instructions. For flow cytometry experiments acquisition was done using either FACSCalibur or FACSCanto II (BD Biosciences). FlowJo Software (TreeStar) was used to analyze cellular events.
5
Immunofluorescence Staining of Cellular Proteins
6
LPS and EORP Stimulation of HASMCs
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HASMCs were plated at a density of 104 cells/well in gelatin-coated coverslips. After starvation, the cells were treated with various concentrations and time of LPS and EORP. After treatment, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then incubated with IL-1β and NF-κB p65 antibodies at 4°C overnight. After incubation, the cells reacted with FITC-conjugated anti-rabbit IgG (both from Sigma) for 1 h at room temperature. DAPI stain was used as the nuclear counterstain and observation was conducted by fluorescence microscope.
7
Immunofluorescence Staining of Cellular Proteins
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Cells were adhered to coverslips and then treated with PEME buffer containing 1% NP-40 for 5 min. The following antibodies were used: FITC-conjugated anti-HA monoclonal antibody (Clone HA-7, 1:400 dilution), anti-HA monoclonal antibody (clone HA-7, 1:400 dilution), and anti-Protein A polyclonal antibody (1:400 dilution). All three antibodies were purchased from Sigma-Aldrich. Cells were incubated with primary antibodies at room temperature for 1 h, and then washed three times with PBS containing 0.1% Triton X-100. For anti-HA and anti-Protein A staining, cells were then incubated with Cy3-conjugated anti-mouse IgG or FITC-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:400 dilution) at room temperature for 1 h. The slides were mounted in VectaShield mounting medium (Vector Labs) containing DAPI and examined using an inverted microscope (Model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60× 1.42-NA DIC objective. Images were acquired and processed using the Slidebook5 software (Intelligent Imaging Innovations, Inc).
8
Immunofluorescent Assay for Poliovirus
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CV1 cells were infected with PV1 (0.01MOI), PV2 (0.05 MOI), or PV3 (0.05MOI), and incubated at 37 °C for 18 h. The cells were fixed with methanol: acetone (1:1) mixture at room temperature for 3 min, and washed twice with PBS. Rabbit anti-VP0, -VP3, -VP1 or -3AB polyclonal sera were added to each well at 1:200 dilution, and incubated at 37 °C for 1 h, and the plates were washed three times with PBS. Then, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma-Aldrich) (1:500 dilution) was added, before incubation at 37 °C for 1 h, and two washes with PBS. The cells were then observed under a fluorescent microscope.
9
Immunocytochemical Analysis of ECFCs
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Control and treated ECFCs were grown on coverslips in EGM-2, fixed and permeabilized according to routine immunocytochemistry methods [18 (link)]. The anti-human primary antibodies used were: anti-uPAR R3 (1:40, rabbit polyclonal, Santa Cruz, CA, USA), anti-caveolin-1 (1:400; Sigma-Aldrich), anti-GM3 (1:100, mouse monoclonal Ab, Cosmo Bio, DBA Italia, Milano, Italy) Cholera toxin-beta subunit (CTB; 10 μg/ml; Sigma-Aldrich) was used to study GM1 distribution. The secondary antibodies used for single and double immunostainings were: CY3-conjugated antimouse IgG (1:800; C2181; Sigma-Aldrich) and FITC-conjugated anti-rabbit IgG (1:800; F-4151; Sigma-Aldrich). For nuclear staining, samples were incubated with DAPI (2 μg/ml), for 15 min. The mounting procedure and the confocal analysis were run as previously described [8 (link)]. Colocalization was determined by ‘Just Another Colocalisation Plugin’ of ImageJ software, as described [9 (link),19 (link)].
10
Immunofluorescence Analysis of H2-Calponin Localization
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For immunofluorescence analysis, cells were cytospun onto slides using a Shandon cytospin III cytocentrifuge and then fixed with 4% paraformaldehyde in phosphate‐buffered saline (PBS) at room temperature for 10 min and perforated with 1% Triton X‐100 in PBS for 4 min. After treatment with 1% bovine serum albumin in PBS for 30 min, the cells were incubated at 4°C overnight with a rabbit anti‐h2‐calponin antibody RAH2 or normal rabbit serum (NS) at 1:200 dilution. A second antibody, FITC‐conjugated anti‐rabbit IgG (Sigma) was utilized at a 1:200 dilution and incubated at room temperature for 60 min in the dark. Filamentous actin was stained with phalloidin–tetramethylrhodamine B isothiocyanate (phalloidin‐TRITC, Sigma) at 1:100 dilution. Coverslips were mounted onto slides with ProLong antifade with DAPI (for nuclear staining) mounting medium (Invitrogen, Carmillo, CA) and air‐dried in the dark for 24 h, sealed with varnish. Cells were examined with a Zeiss Axioimager Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and a 63× , numerical aperture 1.4 oil immersion objective lens. Images were acquired using a Zeiss AxioCam MRm monochrome cooled‐CCD camera and analyzed using Axiovision version 4.8 software (Carl Zeiss).
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