Alexa fluor 488 conjugated goat anti mouse igg

Indirect immunofluorescence assays (IFAs) were performed as previously described [13 (link)]. Cell membranes were permeabilized by 0.2% TritonX-100 in phosphate buffered saline (PBS) for 5 min and blocked in 1% bovine serum albumin (BSA) for 30 min before incubation with primary and secondary antibodies. Primary antibodies against mucin 5B (MUC5B) (1:100 dilution; Abcam, Cambridge, UK) or the zona occludens-1 protein (ZO-1, 1:100 dilution; Abcam) in 1% BSA were incubated with cells overnight at 2–8 °C. Then, cells were washed and incubated for 1 h with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:400, Beyotime Biotech, Nantong, China) or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1:400, Beyotime Biotech). The cells were further stained with 2,4-diamidino-2-phenylindole (DAPI; Beyotime Biotech) for 5 min at room temperature and washed with PBS. Cells were then imaged using an LSM 710 laser scanning confocal microscope (Zeiss, Germany).

The 293T cells were treated with mRNA-containing LNPs, washed three times with PBS, fixed for 20 min with 4% paraformaldehyde, and then permeabilized for 30 min with 0.1% Triton X-100. The fixed cells were incubated with an anti-PEDV-S monoclonal antibody for 2 h at 37°C. After three washes with PBS, the cells were incubated with an Alexa-Fluor-488-conjugated goat anti-mouse IgG (Beyotime) for 1 h at 37°C. The cell nuclei were stained with 0.01% 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. After washing again with PBS, fluorescence images were observed using a fluorescence microscope (Nikon, Japan).

10-d-old red light-grown seedlings were incubated in 50 μM MG132 for 12 h and exposed to 30 μmol m⁻² s⁻¹ blue light for indicated time before nuclear extraction. Nuclear extraction was performed as Yu described with some modifications^{51 (link)}, briefly, seedlings were collected and fixed in 4% formaldehyde for 20 min after blue light treatment, washed twice with PBS, chopped using a razor blade, and suspended in sorting buffer [100 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 0.05% Tween 20, and 5% sucrose], and resuspended with buffer 2[125 mM sucrose,10 mM Tris-HCl, pH 9.5, 10 mM KCl, 0.1%Triton-100], added onto buffer 3[850 mM sucrose,10 mM Tris-HCl, pH 9.5, 10 mM KCl, 0.1%Triton-100) and centrifuged by 15000 rpm at 4 °C for 30 min to precipitate the nuclei. Nuclei were incubated with anti-CRY2 antibody (1:100, prepared in our lab^{52 (link)}) and anti-Myc (1:100, MBL, M192-3) overnight, washed with 0.2%PBST 3 times, incubated with CY3 conjugated goat anti-rabbit IgG (1:500, Beyotime, A0516) and Alexa Fluor 488 conjugated goat anti-mouse IgG (1:500, Beyotime, A0428). Images were captured at 63× oil magnification using a Leica confocal microscope (TCS SP8X, Leica). Immunoblots were used to quantify expression levels of CRY2 and Myc-TCP22. CRY2 and Myc-TCP22 were detected with anti-CRY2 and anti-Myc, respectively.