HEK293T cells were transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector, collected at 36 hpt, fixed with 4% paraformaldehyde overnight at 4 °C, and permeabilized with 0.25% Triton X-100 for 30 min at 4 °C. The cells were rinsed three times with PBST (containing 0.1% Tween-20), blocked with 5% BSA PBS for 2 h at 37 °C, and then incubated with a mouse anti-HA antibody (MBL, Japan, 1:100), goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor 488 (Thermo Fisher Scientific, Meridian Road Rockford, USA, 1:1000). All antibodies were diluted in 1% BSA PBS. Finally, cell nuclei were visualized with DAPI (Roche, Mannheim, Germany). Coverslips were sealed with glycerin buffer, and the cells were visualized using a fluorescence microscope (Nikon ECLIPSE 80i, Japan) [46 (link)].
Goat anti mouse igg h l highly cross adsorbed secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in Japan, China, United Kingdom, United States
Goat anti-Mouse IgG (H + L) highly cross-adsorbed secondary antibody is a laboratory reagent used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays. The antibody is raised in goats and is highly cross-adsorbed to remove non-specific binding to other immunoglobulins, providing specificity for the target mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (2 mentions)
Mouse anti ha antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Anti cd206 antibody, by Abcam (1 mentions)
Goat anti rabbit igg h l highly cross adsorbed secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg conjugated to horseradish peroxidase igg hrp, by Abbkine (1 mentions)
Transzol up, by Transgene (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Triton x 100, by Integra LifeSciences (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Goat serum, by Solarbio (1 mentions)
Triton x 100, by Solarbio (1 mentions)
7 protocols using goat anti mouse igg h l highly cross adsorbed secondary antibody
1
Immunofluorescence Staining of UL41 Proteins
2
Immunohistochemical Staining Panel
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Mouse momoclonal Anti-CD86 antibody, Abcam (Product # 130-122-1), Dilution 1:200; Mouse momoclonal Anti-CD206 antibody, Abcam (Product # 141706), Dilution 1:200; Mouse momoclonal Anti-VEGF antibody, Santa Cruzse (Product # 57496), Dilution 1:200; Mouse momoclonal Anti-EGF antibody, Abcam (Product # EPR19173), Dilution 1:500; Mouse momoclonal Anti-HIF-1 alpha antibody, Abcam (Product # EP1215Y), Use a concentration of 0.5 µg/mL; Mouse momoclonal Anti-CD31 antibody, Abcam (Product # 222783), Dilution 1:1000; Mouse momoclonal Anti-alpha smooth muscle Actin (SMA) antibody, Abcam (Product # ab7817), Dilution 1:500; Mouse momoclonal Anti-VIM antibody, Absin (Product # abs136555), Dilution 1:1000; Mouse momoclonal Anti-COL antibody, Santa Cruzsc (Product # sc-59772), Dilution 1:500; Rabbit polyclonal Anti-Staphylococcus aureus antibody, Absin (Product # ab20920), Dilution 1:1000; Goat Anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, ThermoFisher (Catalog # A-11034), Dilution 1:1000; Goat Anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, ThermoFisher (Catalog # A-21236), Dilution 1:1000.
3
Peptide Screening and Characterization for PEDV
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The peptides were screened by molecular docking in our laboratory by SYBYL-X 2.0 software, synthesized, and purified to at least 90% by GL Biochem (Shanghai, China). The porcine epidemic diarrhea virus S protein (PEDV-S1), PEDV S1 protein monoclonal antibody, anti-his tag mouse monoclonal antibody were made and PEDV N protein monoclonal was conserve in our laboratory, porcine circovirus 2 Cap protein (PCV2-Cap), porcine reproductive and respiratory syndrome virus GP5 protein (PRRSV-GP5) were generated in our laboratory. TransZol Up and PerfectStartIIProbe qPCR SuperMix UDG were purchased from TransGen Biotech (Beijing China). The goat anti-mouse IgG conjugated to horseradish peroxidase (IgG-HRP) was purchased from Abbkine (Wuhan, China) antibody goat anti-mouse IgG(H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488, was purchased by Thermo Fisher Scientific (Invitrogen, Waltham, MA, USA). The cell-counting kit-8 (CCK-8), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and RIPA lysis buffer were purchased from Beyotime (Shanghai, China).
4
Host Responses to Intracellular Pathogens
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Human foreskin fibroblast cells were seeded in 12-well plate and stimulated with 100 U/ml IFN-γ for 24 h, or transfected with 0.5 μg pcDNA3.1-TRIM21-HA for 24 h, or 80 nmol of si-TRIM21 (combining of si-TRIM21-1, si-TRIM21-2, and si-TRIM21-3) or si-NC for 48 h (the sequence of si-TRIM21 are shown in Supplementary Table 2 ), and then stimulated with 100 U/ml IFN-γ for 24 h. After these treatments, the HFF cells were infected with RH tachyzoites (MOI = 3) for 18 h, or CEP tachyzoites (MOI = 3) for 24 h. Subsequently, cell monolayers were subjected to immunofluorescence assay (IFA) to determine the average number of parasites within 100 PVs or the number of PVs containing 1, 2, 4, or 8 tachyzoites from 25 separated fields of view (He et al., 2017 (link)). Anti-SAG1 mouse monoclonal antibody (Abcam, Cambridge, United Kingdom), and Goat anti-Mouse IgG (H + L) highly cross-adsorbed secondary antibody and Alexa Fluor Plus 488 (Invitrogen, United States) were used. Experiments were repeated three times for statistical analysis.
5
Antiviral Screening of Drug Candidates
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Immunofluorescence assay (IFA) was used to further evaluate the antiviral effects of identified drugs. F81 cells in 96-well plates (2.5 × 104 cells/well) were pretreated with 4 μL prediluted Nitazoxanide, Closantel Sodium, and Closantel at final concentrations of 5 μM, 10 μM, and 20 μM, respectively, for 1 h, then treated cells were infected with 10 μL CPV at MOI of 0.076. After 30 h postinfection, cells were fixed with 80% acetone, and then incubated with a 1:100 dilution of mouse anti-VP2 monoclonal antibody (INGENASA, Madrid, Spain) for 40 min, followed by incubation with a 1:200 dilution of fluorescein isothiocyanate-conjugated Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (Invitrogen, Carlsbad, CA, USA). Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) in order to label cell nuclei in focus. After washing, the cells were examined with High Content imaging System (Operetta, PerkinElmer, Waltham, MA, USA) at 20× magnification.
6
Confocal Microscopy Imaging Protocol
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For confocal microscopy imaging, cells were
plated in eight-well chambered slides purchased from ibidi (Gräfelfing,
Germany). Anti-LAMP2 antibody [H4B4]—lysosome marker and normal
goat serum was purchased from Abcam (Waltham, MA). Goat anti-Mouse
IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor
555, and SlowFade Glass (with DAPI) Soft-set antifade mountant were
purchased from Invitrogen (Thermo Fisher Scientific; Waltham, MA).
Gelatin from porcine skin (gel strength ∼ 300 g Bloom) and
BSA were purchased from Sigma (St. Louis, MO). 16% Paraformaldehyde
aqueous solution was purchased from Electron Microscopy Sciences (Hatfield,
PA). Triton X-100 was purchased from Integra (Renton, WA). The Steriflip
vacuum-driven filtration system (0.22 μm) was purchased from
Millipore (Burlington, MA).
plated in eight-well chambered slides purchased from ibidi (Gräfelfing,
Germany). Anti-LAMP2 antibody [H4B4]—lysosome marker and normal
goat serum was purchased from Abcam (Waltham, MA). Goat anti-Mouse
IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor
555, and SlowFade Glass (with DAPI) Soft-set antifade mountant were
purchased from Invitrogen (Thermo Fisher Scientific; Waltham, MA).
Gelatin from porcine skin (gel strength ∼ 300 g Bloom) and
BSA were purchased from Sigma (St. Louis, MO). 16% Paraformaldehyde
aqueous solution was purchased from Electron Microscopy Sciences (Hatfield,
PA). Triton X-100 was purchased from Integra (Renton, WA). The Steriflip
vacuum-driven filtration system (0.22 μm) was purchased from
Millipore (Burlington, MA).
7
SCAP Localization in Golgi Complex
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The treated cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS with 0.05% Tween-20 (SigmaAldrich) for 5 min. Then cells were permeabilized with 0.02% TritonX-100 (Solarbio) and blocked with goat serum (Solarbio) for 1 h. All the cells were incubated with rabbit anti-human SCAP antibody (1:100, Cat. No. ab190103, Abcam) and mouse anti-Golgi antibody (1:100, Cat. No. A-21270, Invitrogen) for 12 h at 4°C. After washing three times using PBS/Tween-20 over 30 min, cells were dual-stained with goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (green) for SCAP (1:100, Cat. No. A-11034, Invitrogen) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (red) for Golgin (1:100, Cat. No. A-11032, Invitrogen) for 1 h at room temperature. Finally, cells were observed under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany).
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