Abflo 647 conjugated goat anti rabbit igg h l

To investigate the colocalization of RACK1, NLRP3 and NEK7, cells were prepared as described above and transfected with the pcDNA3.1-eGFP-NEK7 plasmid for 48 h. Then, the cells were infected with PmCQ2 for 3 or 4 h. After infection, the cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT). After three wash steps, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at RT. After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight. Next, secondary Abs including goat anti-mouse IgG (H&L) Alexa Fluor 488 (Abcam, UK), goat anti-rabbit IgG (H&L) Alexa Fluor 594 (Abcam, UK) and ABflo™ 647-conjugated goat anti-rabbit IgG (H&L) (Abclonal, China) were added after washing with PBS and incubated for 1 h at RT. Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubated in the dark for 5 min. Finally, anti-fluorescence attenuation mounting tablets (Solarbio, Beijing, China) were used, and the results were observed using fluorescence microscopy (Olympus, Tokyo, Japan).

IF assays were performed as previously described (Zhu et al. 2021 (link)). Briefly, treated cells were washed, fixed, permeabilized, and blocking. Subsequently, the cells were co-incubated overnight with an anti-NEDD4 (21698-1-AP, Proteintech, China) antibody at 4 °C, followed by three washes with PBS. Next, the cells were incubated for one hour with ABflo 488-conjugated Goat Anti-Rabbit IgG (H + L) (AS053, ABclonal, China), then washed three times with PBS. The cells were then incubated overnight with the anti-ECHDC2 (bs-13049R, Bioss, China) antibody at 4 °C, followed by washing with PBS. Afterward, the cells were incubated for one hour with ABflo 555-conjugated Goat Anti-Rabbit IgG (H + L) (AS058, ABclonal, China), then washed three times with PBS, followed by overnight incubation with the anti-MCCC2 (12117-1-AP, Proteintech, China) antibody at 4 °C, and subsequent PBS washing. Finally, the cells were incubated for one hour with ABflo 647-conjugated Goat Anti-Rabbit IgG (H + L) (AS060, ABclonal, China), and stained for 15 min with DAPI (Cell Signaling Technology, USA). Imaging of stained cells was captured using a Zeiss LSM 900 confocal microscope.