ST cells inoculated with TGEV were cultured for 24 h. The cells were washed twice with PBS and fixed with paraformaldehyde (4%) for 30 min at 4 °C, and then allowed to air dry. After blotting with 5% skimmed milk powder, the fixed cells were incubated with mAb to TGEV N protein (1:100) and rabbit pAb to EF1A (1:50) for 1 h at 37 °C in a humidified chamber. After washing three times with PBST, the fixed cells were incubated with FITC-labeled goat anti-mouse IgG (1:100, KPL) and TRITC-labeled goat anti-rabbit IgG (1:200, Sigma). The additional nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was performed as described previously (Jungmann et al., 2001 (link)). The triple-stained cells were washed three times with PBST and subsequently examined under a Leica TCS SP5 laser confocal microscopy.
Tritc labeled goat anti rabbit igg
Manufactured by Merck Group
Sourced in United States
TRITC-labeled goat anti-rabbit IgG is a secondary antibody that binds to rabbit immunoglobulin G (IgG). The antibody is conjugated with the fluorescent dye TRITC (Tetramethylrhodamine), which allows for visualization of target proteins in various applications such as immunofluorescence and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Tcs sp5 laser confocal microscopy, by Leica (2 mentions)
Ab9484, by Abcam (2 mentions)
Tcs sp5 confocal laser microscope, by Leica (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab140632, by Abcam (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
6 protocols using tritc labeled goat anti rabbit igg
1
Immunofluorescence Analysis of TGEV Infection in ST Cells
2
Visualization of TGEV Infection in PK-15 Cells
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PK-15 cells infected with TGEV H strain (MOI of 0.1) were cultured for 36 h. The cells were fixed and blocked as described above and incubated with the primary antibody (mAb 5E8, 1:100) for 60 min at 37°C, after which they were washed three times with PBST. Subsequently, the cells were incubated with secondary antibody (fluorescein isothiocyanate [FITC]-labeled goat anti-mouse IgG, Kirkegaard & Perry, Gaithersburg, MD, USA). The nucleolus was visualized using a primary antibody (rabbit polyclonal antibody to nucleolin, 1:100, Abcam); the secondary antibody used was tetramethylrhodamine (TRITC)-labeled goat anti-rabbit IgG (1:200, Sigma). Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI, Sigma) was performed as previously described [19 (link)]. The stained cells were washed three times with PBST and subsequently examined under a Leica TCS SP5 laser confocal microscope.
3
Antibody Labeling and TGEV Detection
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Mouse mAb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9484) and Rabbit poloclonal antibody vimentin (ab92547) were purchased from Abcam. FITC-labeled goat anti-mouse IgG was purchased from Kirkegaard and Perry Laboratories (KPL). TRITC-labeled goat anti-rabbit IgG was purchased from Sigma. The mAb to N protein of TGEV were donated from Mr. Wang Shao (Institute of Animal Husbandry and Veterinary Science, Fujian Academy of Agricultural Science, China).
4
Immunofluorescence Antibody Detection
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Mouse monoclonal antibody (mAb) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9484) and rabbit polyclonal antibody (pAb) to EF1A (ab140632) were purchased from Abcam. FITC-labeled goat anti-mouse IgG was purchased from Kirkegaard and Perry Laboratories (KPL). TRITC-labeled goat anti-rabbit IgG was purchased from Sigma mAb to N protein of TGEV was prepared in our lab.
5
Visualizing TGEV Infection in ST Cells
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ST cells inoculated with TGEV were cultured for 0, 1, 2, 4, 8, and 16 h. Cells were washed twice with PBS and fixed with paraformaldehyde (4%) for 30 min at 4 °C, and then allowed to air dry. After blotting with 5% skimmed milk powder, the fixed cells were incubated with mAb to TGEV N protein (1:200) and rabbit pAb to vimentin (1:100, Abcam) for 1 h at 37 °C in a humidified chamber. After washing three times with PBST, the fixed cells were incubated with FITC-labeled goat anti-mouse IgG (1:100, KPL) and TRITC-labeled goat anti-rabbit IgG (1:200, Sigma). Additional nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was performed as described previously (Jungmann et al., 2001 (link)). The triple-stained cells were washed three times with PBST and subsequently examined under a Leica TCS SP5 laser confocal microscopy.
6
Comparative Analysis of PEDV Isolates
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To compare the propagation effects of PEDV isolates 2013-A and NJ in Vero, Vero/TMPRSS2, and Vero/MSPL cells, an immunofluorescence assay was performed according to the methods described previously with slight modifications [38 ]. Vero/TMPRSS2, Vero/MSPL, and Vero cells were seeded in 12-well plates and incubated with PEDV isolates at MOI = 0.01 and cultured with or without trypsin as described above. After washing and fixing with 4% paraformaldehyde, and permeabilization with 0.2% Triton X-100, the cells were treated with rabbit anti-PEDV polyclonal antibody (dilution at 1:200) as primary antibody and TRITC-labeled goat anti-rabbit IgG (Sigma, St. Louis, MO, USA) as secondary antibody followed by the counterstain of nuclei with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, USA), fluorescence images were obtained by a fluorescence microscope (Leica, Wetzlar, Germany).
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