Anti fluorescence attenuation mounting tablets

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).

Cells were prepared as described and infected with PmCQ2 for 3 or 4 h. After infection, cells were fixed with 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT) and then blocked with 5% Bovine Serum Albumin (BSA) for 1 h at RT. After washing steps, primary antibodies (anti-ASC Ab, Santa Cruz, CA; anti-NLRP3 Ab, Wanlei Life Sciences, Shenyang, China) were added and incubated overnight at 4°C. Next, Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, United Kingdom) was added after washing with PBS at RT for 1 h. 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 an inverted fluorescence microscope (Olympus, Tokyo, Japan).
To show the extent of speck formation more intuitively, the percentage of cells that contained a speck was determined. Cells from five different fields (average of 100 cells/field) were counted based on DAPI-stained nuclei for each of different experiments. Images were analyzed using ImageJ. The data is expressed as the percentage of cells with specks per number of cells per field.