HepG2 cells (4×104) were seeded onto eight-well Lab-Tek II CC2 slides (Nunc) one day before infection. IFA for detection of HEV-infected cells was performed as described [62 (link)]. To examine the subcellular localization of MAVS and IRF3, cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.2% Triton X-100 for 15 minutes. Cells were stained with pre-absorbed ch1313 serum and a rabbit anti-MAVS, mouse anti-PMP70, or rabbit anti-IRF3 for 1 h, and subsequently incubated with Alexa Fluor 488/594-conjugated goat-anti-rabbit IgG, Alexa Fluor 488/594-conjugated goat-anti-mouse IgG, or Alexa Fluor 488-conjugated goat-anti-human IgG (Invitrogen) for 1 h. After adding antifade-4 6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), slides were viewed with a Zeiss LSM 510 confocal microscope with a 63x (NA1.2) apochromatic water objective. Images were acquired using the ZEN 2009 software.
Antifade 4 6 diamidino 2 phenylindole dapi mounting solution
Manufactured by Merck Group
Antifade-4 6-diamidino-2-phenylindole (DAPI) mounting solution is a laboratory reagent used to stain and preserve nucleic acids. It is a fluorescent dye that binds strongly to DNA, allowing for the visualization of cell nuclei in microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 cc2 slides, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Smartpool sirna, by Horizon Discovery (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Celigo imaging cytometry system, by Revvity (1 mentions)
Dm ire2 confocal microscope, by Leica (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti hemagglutinin ha antibody, by Merck Group (1 mentions)
3 protocols using antifade 4 6 diamidino 2 phenylindole dapi mounting solution
1
Immunofluorescence Assay for HEV Infection
2
SARS-CoV-2 Infection Assay in Calu-3 Cells
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The siRNA transfection was conducted in 384-well plate format. 6,000 Calu-3 cells were plated and transfected with siRNA SMARTpools (Dharmacon) using RNAiMAX (Invitrogen). 48 h post-transfection, cells were infected with SARS-CoV-2 at MOI of 0.125 per well. 48 h post-infection, infected cells were fixed with 5% paraformaldehyde (PFA) for 4 h and permeabilized with 0.4% Triton X-100 for 15 min. After blocking with blocking buffer containing 3% bovine serum albumin (BSA) and 10% goat serum for 30 min, cells were incubated for overnight at 4°C with rabbit-anti-SARS-CoV-2 NP polyclonal antibodies. After three washes with phosphate-buffered saline (PBS), the cells were incubated with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (Thermo Fisher Scientific, USA) for 1 h at room temperature. After three additional washes, antifade-4 6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma) was added to the cells for 30 min before imaging. The fluorescence intensity of cells was scanned and the SARS-CoV-2 NP-positive cells were automatically calculated by Celigo imaging cytometry system (Nexcelom, Lawrence, MA, USA) or using the IC200 imaging system (Vala Sciences, CA, USA).
3
Visualizing Tetherin Localization during Viral Infection
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Localization experiments were performed using IFAs and laser scanning confocal microscopy. Briefly, MDCK cells grown on glass coverslips were transiently transfected with the appropriate tetherin expression constructs by employing Lipofectamine® 3000. At 24 h after transfection, the cells were infected with CIV H3N2 at an MOI of 0.1, as described previously. At 48 h after infection, the cells were fixed with 4% paraformaldehyde-PBS. The cells were incubated with an anti-hemagglutinin (HA) antibody (Sigma, Saint Louis, MO, USA) to detect the HA protein and with anti-FLAG antibody (Sigma, Saint Louis, MO, USA) to detect canine tetherin and then they were stained using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) and cyanine-3 (Cy3). After mounting on slides using antifade 4′,6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), the cells were visualized with a Leica DM-IRE2 confocal microscope.
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