MRC5 cells were grown to confluence in a 96-well dish and infected with mock or OC43 at an MOI of 0.01 or 1. At 48 h post infection, cells were washed 3x with PBS and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed 3x with PBS and permeabilized with PBS containing 0.1% TritonX-100 for 15 min at room temperature. Cells were blocked with PBS containing 2% BSA and 0.05% Tween-20 for 1 h at room temperature. N-protein antibody (Sino Biological #40068) was prepared to 1:1000 dilution in PBS containing 0.5% Tween-20 and added to each well for 1 h. Cells were washed 3x with PBS containing 0.01% Tween-20. Secondary antibody (alexa flour 594 goat-anti-rabbit IgG, Invitrogen #a-11012) was prepared to 1:1000 in PBS containing 0.5% Tween-20 and added to each well for 1 h. Cells were washed 3x with PBS containing 0.05% Tween-20, Hoeschet stain (1:1000) was added to the final wash of PBS. Cells were imaged using the Cytation 5 imaging reader (BioTek). Each well was imaged using a 20X magnification objective lens with a predefined DAPI channel (described above) and predefined Texas Red channel with an excitation wavelength of 586 nm and emission wavelength of 603 nm.
Alexa flour 594 goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594 goat anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti transgelin, by Abcam (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Alexa flour 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Pcdna6 myc his b taglnag plasmid, by Takara Bio (2 mentions)
Pcdna6 myc his b ag plasmid, by Takara Bio (2 mentions)
Penter flag control plasmid, by Charles River Laboratories (2 mentions)
Cytation 5 imaging reader, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Gtx125932, by GeneTex (1 mentions)
Mouse monoclonal anti flag m2 antibody f1804, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
63 oil immersion objective lens, by Zeiss (1 mentions)
Anti parp1, by Cell Signaling Technology (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
7 protocols using alexa flour 594 goat anti rabbit igg
1
Quantitative SARS-CoV-2 N-protein Immunoassay
2
Indirect Immunofluorescence Staining of Influenza Proteins
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For indirect immunofluorescence staining, cells were fixed with 4% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 1% BSA for 60 min, and then probed with the indicated primary antibodies, including mouse monoclonal anti-IAV NP antibody (1331, 1:200, ViroStat, Portland, ME, USA), rabbit anti-human galectin-3 antibody (sc-20157, 1:200, Santa Cruz), rabbit anti-influenza A PA antibody (GTX125932, 1:500, GeneTex), and mouse monoclonal anti-Flag M2 antibody (F1804, 1:500, Sigma-Aldrich), overnight at 4 °C. Alexa Flour 488-goat anti-mouse IgG (A-21202, 1:200, Invitrogen) and Alexa Flour 594-goat anti-rabbit IgG (A-11012, 1:200, Invitrogen) were used as secondary antibodies. The nuclei were counterstained with DAPI (Sigma-Aldrich). Images were acquired using an Olympus FV1000 confocal microscope or observed under fluorescence microscopy (Olympus, Tokyo, Japan).
3
Co-Immunostaining of FFPE Tissue Sections
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We performed co-immunostaining for FFPE sections as described in previous publications [10 (link),24 (link)]. Briefly, we performed deparaffinization, hydration, antigen retrieval and blocking of endogenous peroxidase activity and non-specific binding as described in the immunohistochemical staining section above. We prepared the primary antibodies in 5% goat serum as follows: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Tie2 1:400 (Invitrogen, Burlington, ON, Canada, # PA5-28582). The sections were incubated with the primary antibodies at 4°C overnight. Next day, the sections were washed and incubated with a mixture of secondary antibodies for 2 h. The secondary antibody mixture was composed of 5% goat serum containing the following antibodies: Alexa Flour 594 goat anti-rabbit IgG 1:500 (Invitrogen, Burlington, ON, Canada, #A11037) and Alexa Flour 488 goat anti-mouse IgG 1:500 (Invitrogen, Burlington, ON, Canada, #A10680). Next, the sections were washed and incubated with DAPI (1:1000 in 1× PBS) for 10 min. The sections were then protected with coverslips using ProLong Mountant (Thermo Fisher Scientific, Saint-Laurent, QC, Canada, #P36934).
4
Indirect Immunofluorescence of Viral Proteins
5
Localization and Expression of Cytoskeletal and Nuclear Proteins
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Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines and the expression of PARP1 in RKO cells were determined by immunofluorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA; anti-PARP1, 1:500, Cell signaling technology, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, Alexa Flour 488 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′, 6-diamidino-2-phenylindole (DAPI) were used. The immunofluorescence images were taken and preserved under the laser scanning confocal microscope using a 63 × oil-immersion objective lens (Carl Zeiss, USA).
6
Subcellular localization of transgelin in colorectal cancer cells
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Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines was determined by immuno uorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′,6-diamidino-2-phenylindole (DAPI) were used. The immuno uorescence images were taken and preserved under the laser scanning confocal microscope (63× oil lens, Carl Zeiss, USA). Transfection SW480 and RKO cells were cultured in 12-well plates and transfected with pcDNA6/myc-His B-TAGLNag plasmid and pcDNA6/myc-His B-ag plasmid (Takara, Japan). In the validation experiment, we transfected the RKO cells with pENTER-TAGLN-Flag and pENTER-Flag control plasmid (Vigene Biosciences, USA). Transfection was conducted using Lipofectamine 2000/ Lipofectamine 3000 (Thermo Fisher Scienti c, USA). The cells were harvested at 48 hours after transfection for further analysis.
7
Localization and Transfection of Transgelin
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Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines was determined by immuno uorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′,6-diamidino-2-phenylindole (DAPI) were used. The immuno uorescence images were taken and preserved under the laser scanning confocal microscope (63 × oil lens, Carl Zeiss, USA). Transfection SW480 and RKO cells were cultured in 12-well plates and transfected with pcDNA6/myc-His B-TAGLNag plasmid and pcDNA6/myc-His B-ag plasmid (Takara, Japan). In the validation experiment, we transfected the RKO cells with pENTER-TAGLN-Flag and pENTER-Flag control plasmid (Vigene Biosciences, USA). Transfection was conducted using Lipofectamine 2000/ Lipofectamine 3000 (Thermo Fisher Scienti c, USA). The cells were harvested at 48 hours after transfection for further analysis.
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