For immunofluorescent staining, the retinas were permeated and blocked with 1% Triton X-100 in 1% bovine serum albumin (BSA) for 1 h at room temperature (RT) after separation. After that, the retinas were stained with primary antibody CD11b (Serotec, Oxford, UK), Iba-1 (Wako Chemicals, Osaka, Japan), interleukin-1 beta (IL-1β; Abcam, Cambridge, MA), and chemokine (C-C motif) ligand 2 (CCL-2; Novus Biologicals, Littleton, CO) for 18 h at 4 °C. After washing with PBS, the retinas were incubated with the appropriate secondary antibodies conjugated with fluorescent dye (Alexa Fluor 555 or 488, Cell Signaling, Danvers, MA) at RT for 1 h. Finally, the retinas were flat-mounted and examined with a confocal microscope.
Alexa fluor 555 or 488
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor® 555 and 488 are fluorescent dyes used in various biomedical applications. They exhibit high brightness and photostability, making them suitable for a range of imaging techniques.
Automatically generated - may contain errors
3 protocols using alexa fluor 555 or 488
1
Immunofluorescent Staining of Retinal Tissues
2
Immunofluorescence Analysis of p65 and PRRSV nsp2
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Marc-145 cells (8 × 105 cells/well) were seeded in 12-well plates or confocal Petri dishes. Cells were fixed in 4% paraformaldehyde for 15 min. After permeabilization with 0.5% Triton X-100 for 15 min at room temperature (RT), cells were blocked with 1% bovine serum albumin (BSA) for 30 min and then incubated with a rabbit monoclonal antibody against the p65 protein (1:500 dilution, Cell Signaling Technology) or an antibody against PRRSV nsp2 (a gift from Dr. Hanchun Yang, China Agricultural University, 1:1000) at 4 °C overnight. Cells were then incubated with an anti-rabbit secondary antibody conjugated with Alexa Fluor® 555 or 488 (Cell Signaling Technology, MA, USA) at 1:1000 dilution for 2 h. Nuclei were stained using DAPI (1:1000; Cell Signaling Technology). Cells were examined by fluorescence microscopy (Nikon, Tokyo, Japan).
3
Immunofluorescence Analysis of p65 and PRRSV
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Cells were xed with 4% paraformaldehyde for 10 min. After permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT), cells were blocked with 1% bovine serum albumin (BSA) for 30 min at RT and then incubated with a rabbit monoclonal antibody against the p65-protein (1:500 dilution, Cell
Signaling Technology) or an antibody against PRRSV nsp2 (a gift from Dr. Hanchun Yang, China Agricultural University, 1:1000) at 4 °C overnight. After three washes with PBS, the cells were incubated for 1 h at RT with an anti-rabbit secondary antibody conjugated with Alexa Fluor® 555 or 488 (Cell Signaling Technology, MA, USA) at 1:1000 dilution. Nuclei were counterstained using DAPI (1:1000; Cell Signaling Technology). Cells were examined by uorescence microscopy (Nikon, Japan).
Signaling Technology) or an antibody against PRRSV nsp2 (a gift from Dr. Hanchun Yang, China Agricultural University, 1:1000) at 4 °C overnight. After three washes with PBS, the cells were incubated for 1 h at RT with an anti-rabbit secondary antibody conjugated with Alexa Fluor® 555 or 488 (Cell Signaling Technology, MA, USA) at 1:1000 dilution. Nuclei were counterstained using DAPI (1:1000; Cell Signaling Technology). Cells were examined by uorescence microscopy (Nikon, Japan).
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