To analyze the colocalization of ISG15 with PPRV proteins, EECs after cotransfection with plasmids or infection with PPRV were fixed with 4% paraformaldehyde for 30 min at room temperature and washed three times with PBS for 6 min each. The cells were permeabilized by prechilled methanol at −20°C and incubated at the same temperature for 10 min. The cells were blocked with 5% bovine serum albumin and 0.3% Triton X-100 at room temperature for 2 h. Primary antibodies and secondary antibodies (Alexa Fluor 594 goat anti-rabbit immunoglobulin G [H+L] and Alexa Fluor 488 goat anti-mouse immunoglobulin G [H+L]; Invitrogen) were then added. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were taken with a Zeiss LSM880 confocal microscope (Zeiss).
Alexa fluor 488 goat anti mouse immunoglobulin g h l
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 goat anti-mouse immunoglobulin G [H+L] is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize mouse immunoglobulin G (IgG) proteins in various immunological and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 goat anti rabbit immunoglobulin g h l, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
4 protocols using alexa fluor 488 goat anti mouse immunoglobulin g h l
1
Colocalization of ISG15 and PPRV Proteins
2
Colocalization of ISG15 and PPRV Proteins
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To analyze the colocalization of ISG15 with PPRV proteins, EECs after cotransfection with plasmids or infection with PPRV were fixed with 4% paraformaldehyde for 30 min at room temperature and washed three times with PBS for 6 min each. The cells were permeabilized by prechilled methanol at −20°C and incubated at the same temperature for 10 min. The cells were blocked with 5% bovine serum albumin and 0.3% Triton X-100 at room temperature for 2 h. Primary antibodies and secondary antibodies (Alexa Fluor 594 goat anti-rabbit immunoglobulin G [H+L] and Alexa Fluor 488 goat anti-mouse immunoglobulin G [H+L]; Invitrogen) were then added. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were taken with a Zeiss LSM880 confocal microscope (Zeiss).
3
Immunofluorescent Staining of CyHV-3 Proteins
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Cells were fixed in PBS containing 4% (w/v) paraformaldehyde at 4°C for 15 min and then 20°C for 30 min. After washing with PBS, samples were permeabilized in PBS containing 0.1% (v/v) NP-40 at 37°C for 15 min. Immunofluorescent staining (incubation and washes) was performed in PBS containing 10% FCS (v/v). Mouse pAb raised against pORF56 (diluted 1:500), mAb 6B2 raised against pORF57 (diluted 1:2500), and rabbit pAb raised against CyHV-3 structural proteins (diluted 1:1500) were used as the primary antibodies. The primary antibody was incubated at 37°C for 1 h. Alexa Fluor 488 goat anti-mouse immunoglobulin G (H+L) and Alexa Fluor 568 goat anti-rabbit immunoglobulin G (H+L) (Invitrogen) were used as the secondary antibodies. The secondary antibody was incubated at 37°C for 30 min. After washing, cells were mounted using Prolong Gold Antifade Reagent with DAPI (Invitrogen).
4
Immunofluorescent staining of MNV1-infected cells
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RAW 247.6 cells were plated on 18 mm glass coverslips in 12-well plates and grown overnight. At a confluency of 70%, cells were infected with MNV1-CW1 at a MOI of 0.1. Twenty hours later, cells were fixed in 4% (v/v) paraformaldehyde in PBS for 25 min. After washing with PBS, samples were permeabilised in PBS containing 0.1% (v/v) Triton X-100 at room temperature for 15 min. Immunofluorescent staining (incubation and washes) was performed in PBS containing 10% FCS (v/v) and 0.1% BSA. Antipeptide antibodies were used as the primary antibody at dilution 1:500 (R2 sera) and 1:200 (R12 sera) for 1 h at room temperature, followed by Alexa Fluor 488 goat anti-mouse immunoglobulin G (H + L) (Invitrogen) as the secondary antibody (1:1000 dilution). After washing, cells were mounted using Prolong Gold antifade reagent with Di Aminido Phenyl lndol (DAPI) (Invitrogen). Samples were analysed by confocal microscopy, using a Leica SP5 confocal microscope and compiled using ImageJ Software (Schindelin et al., 2012) (link).
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