Enteroid monolayers grown in 96-well plates were respectively infected with the three SECoVs for 24 h. Enteroid monolayers were fixed with 4% paraformaldehyde (PFA) for 30 min at RT, permeabilized with PBS containing 0.2% Triton X-100 for 20 min, and then blocked with 5% FBS and 5% skim milk (Sigma-Aldrich, USA) diluted in PBS at 37°C for 2 h. The cells were respectively incubated with nucleocapsid (N) antibody of PEDV, TGEV, or PDCoV overnight at 4°C, and then labeled with Alexa Fluor 546 goat anti-mouse IgG antibody (Thermo Fisher Scientific, USA) at 37°C for 1 h. DAPI (4’,6-diamidino-2-phenylindole) was used to stain nuclear DNA. The cells were imaged using an Evos FL Auto2 fluorescence microscope.
Alexa fluor 546 goat anti mouse igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 546 goat anti-mouse IgG antibody is a fluorescently-labeled secondary antibody used for detecting and visualizing mouse primary antibodies in various immunodetection applications, such as immunofluorescence and flow cytometry. The Alexa Fluor 546 dye provides bright, photostable fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Alexa fluor 488 goat anti rabbit igg antibody, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Anti ha monoclonal antibody, by Merck Group (2 mentions)
Alexa fluor 546 goat anti rabbit igg antibody, by Thermo Fisher Scientific (2 mentions)
Skim milk, by Merck Group (1 mentions)
Tcs sp5 confocal microscopy, by Leica camera (1 mentions)
Emeraldamp pcr master mix, by Takara Bio (1 mentions)
Dmem with high glucose, by Merck Group (1 mentions)
Dl2000 dna marker, by Tiangen Biotech (1 mentions)
Tiangel midi purification kit, by Tiangen Biotech (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Rabbit anti gfp antibody, by Takara Bio (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
13 protocols using alexa fluor 546 goat anti mouse igg antibody
1
Enteroid Monolayer Infection with SECoVs
2
PEDV Infection Inhibition Assay
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Vero E6 or IPEC-J2 cells were seeded in 96-well plates, and each confluent monolayer of cells was stimulated with indicated concentrations of either type of rpIFN-L for 24 h before PEDV infection. The cells were then infected with PEDV strain CV777 at an MOI of 0.1. PEDV infection was analyzed using an immunofluorescence assay (IFA) at 36 h post-infection. Briefly, the cells were fixed with 4% paraformaldehyde at 4 °C for 30 min and washed with 1 × PBS. Fixed cells were permeabilized with 0.2% Triton X-100 for 15 min at room temperature and blocked with blocking buffer (PBS with 10% FBS) for 1 h. The preparations were labeled with the mouse anti-PEDV nucleocapsid monoclonal antibody (mAb) 2G3 stocked in our laboratory (1:100 dilution) at 37 °C for 2 h followed by labeling with the Alexa Fluor 546 goat anti-mouse IgG antibody (1:200 dilution) (ThermoFisher Scientific) for 1 h at 37 °C. DAPI (1:100 dilution) was used to stain cell nuclei. The stained cells were visualized using an AMG EVOS F1 florescence microscope.
3
Immunostaining of PPV-Infected Cells
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To determine whether the isolated virus binds the PPV monoclonal antibody, the PPV-infected ST cells were fixed with 4 % paraformaldehyde for 30 min and then permeabilized with 0.2 % Triton X-100 for 20 min. The cells were stained with mouse anti-PPV capsid protein (VP2) monoclonal antibody (1B7 mAb) at 37 °C for 1 h. The bound antibodies were visualized using Alexa Fluor 546 goat anti-mouse IgG antibody (1:200 dilution) (Thermo Fisher Scientific) for 45 min at 37 °C. Cell nuclei were counterstain with DAPI (1 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were analyzed using an AMG EVOS F1 fluorescence microscope and Leica TCS sp5 confocal microscopy.
4
Isolation and Characterization of PPV-BQ Strain
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The PPV-BQ strain (GenBank: EU790641.1) was isolated, cultivated, and preserved by the innovation team of pig digestive tract infectious diseases of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The porcine testis cell line (ST) and mouse anti-PPV capsid protein (VP2) monoclonal antibody (1B7 mAb) were prepared and preserved by our laboratory; DMEM with high glucose was purchased from Sigma (China); 0.25 % trypsin-EDTA (1 ×) and RPMI Medium 1640 basic (+ L -Glutamine) were purchased from Gibco; Special fetal bovine serum was purchased from Inner Mongolia Opcel Biotechnology Co., Ltd.; TIANamp Genomic DNA Kit and TIANgel Midi Purification Kit were purchased from Tiangen; DL 2000 DNA Marker, DH5α Competent cell, pMD18-T Vector, and EmeraldAmp PCR Master Mix (2 × Premix) were purchased from TaKaRa (Dalian, China); SafeViewTM FireRed was purchased from ABM; T4 DNA ligase was purchased from NEB (Beijing, China); the 0.22 μm filter was purchased from Merck Chemicals (Shanghai) Co., Ltd.; and AlexaFluor 546 goat anti-mouse IgG antibody (1:200 dilution) was purchased from Thermo Fisher Scientific (China).
5
Immunofluorescence Staining of Vero E6 Cells
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Vero E6 cells were fixed with 4% paraformaldehyde for 30 min at 4°C and permeabilized with 0.2% Triton X-100 for 15 min, then blocked with blocking buffer (PBS with 5% FBS) for 2 h at 37°C. The cells were incubated with an anti-HA monoclonal antibody (Sigma-Aldrich, Munich, Germany, 1:5000) at 37°C for 2 h, followed by labeling with an Alexa Fluor 546 goat anti-mouse IgG antibody (Thermo Fisher Scientific, 1:500) at 37°C for 1 h. 4′,6-diamidino-2-phenylindole (DAPI, 1:100) was used to stain the cellular nuclei. The stained cells were visualized using an AMG EVOS F1 fluorescence microscope.
6
Immunofluorescence Assay for Viral Protein Detection
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Cell samples collected as described previously were washed with PBS two times, fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. PBS containing 5% serum and nonfat dry milk was used as blocking buffer overnight at 4°C, incubated with monoclonal antibodies against TGEV N protein (1:1,000), PDCoV S protein (1:1,000), or anti-HA antibody (1:5,000) for 2 h at 37°C. After three washings with PBST (PBS with 0.05% Tween 20), samples were stained for 1 h with Alexa Fluor 546 goat anti-mouse IgG antibody or Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500; Thermo Fisher Scientific, USA) at 37°C. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:100; Sigma, USA) and then visualized using an Evos FL Auto2 fluorescence microscope.
7
PEDV N Protein Immunofluorescence Assay
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IPEC-J2 or Vero E6 cells were fixed with 4% paraformaldehyde for 30 min at RT. The fixed cells were permeabilized with 0.2% Triton X-100 for 20 min at RT and then blocked with blocking buffer (PBS with 5% FBS and 5% skim milk) for 30 min at 37°C. The cells were then incubated with PEDV N protein antibody at 37°C for 2 h and then labeled with Alexa Fluor 546 goat anti-mouse IgG antibody (Thermo Fisher Scientific, USA) at 37°C for 1 h. DAPI (Sigma, USA) was used for the staining of cellular nuclei. The stained cells were visualized using an AMG EVOS F1 fluorescence microscope (Thermo Fisher Scientific, USA).
8
Immunofluorescence Staining of ST Cells
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ST cells were fixed with 4% paraformalde for 30 min at 4 °C, permeabilized with 0.2% Triton X-100 for 15 min at room temperature (RT), and blocked with blocking buffer (PBST with 5% milk) for 30 min at 37 °C. The cells were then incubated with anti-HA monoclonal antibody (Sigma-Aldrich) or TGEV N protein antibody at 37 °C for 2 h, followed by labeling with Alexa Fluor 546 goat anti-mouse IgG antibody (Thermo Fisher Scientific) at 37 °C for 1 h. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). The stained cells were visualized using an AMG EVOS F1 fluorescence microscope.
9
Immunostaining of cultured Plasmodium parasites
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Smears of cultured PyXNL parasites were fixed on glass slides with ice-cold acetone for 5 min and blocked with PBS containing 5% nonfat milk at 37 °C for 30 min. They were then incubated with rabbit anti-CryPH antibodies (1: 1000), rabbit anti-GFP antibody (1: 500, Clontech, Mountain View, CA, USA) and mouse anti-Pys25 mAb (1: 20,000) at 37 °C for 1 h, and thereafter with Alexa Fluor 488-goat anti-rabbit IgG antibody and Alexa Fluor 546-goat anti-mouse IgG antibody (Invitrogen) as secondary antibodies (1:500 dilution) at 37 °C for 30 min, together with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). After mounting in ProLong Gold antifade reagent (Invitrogen), samples were observed with an inverted fluorescence microscope (Axio observer z1, Carl Zeiss, Oberkochen, Germany), and images were taken using AxioVision software (Carl Zeiss).
10
Molecular Mechanisms in Inflammatory Response
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Angiotensin II and JSH-23 were purchased from Sigma-Aldrich (St. Louis, MO). The primary antibodies were obtained from the following sources: anti-LC3, anti-p-p65 NF-κB (Ser536), and anti-p65 NF-κB (Cell Signaling Technology, Beverly, MA), anti-ATG5, anti-IL-1β, anti-F4/80, anti-CD3, and anti-p-H3 (Abcam, Cambridge, MA); Anti-p62 GeneTex (Irvine, CA); anti-FLAG and anti-HA (Invitrogen Life Technologies, Paisley, UK); Alexa Fluor 488 goat anti-rabbit IgG antibody, Alexa Fluor 488 goat anti-mouse IgG antibody, Alexa Fluor 546 goat anti-mouse IgG antibody, and Alexa Fluor546 goat anti-rabbit IgG antibody (Invitrogen Life Technologies, Paisley, UK).
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