Immunostaining permeabilization solution with triton x 100

TM4 cells cultured on coverslips were fixed in 4% paraformaldehyde in PBS (10 mM sodium phosphate and 0.15 M NaCl, pH 7.4, at 22 °C; w/v) for 10 min and permeabilized in Immunostaining Permeabilization Solution with Triton X-100 (Beyotime Ltd., Zhejiang, China) for 5 min. Cells were then blocked with 5% goad serum in PBS (v/v; blocking solution), followed by overnight incubation at 4 °C with primary antibodies diluted in blocking solution: mouse anti-p62 (Proteintech; 1:100). Thereafter, cells were washed in PBS and incubated with secondary fluorescent antibody Alexa Fluor–conjugated secondary antibodies (Abcam, Cambridge, UK; red fluorescence, Alexa Fluor 555) at 1:250 dilution with blocking solution at room temperature for 1h. For mitochodrion staining, cells were only incubated with Mito-Tracker Red CMXRos (Beyotime, 1:1000) diluted in culture medium. Cells were then washed and mounted with Antifade Mounting Medium with 4-,6-diamidino-2-phenylindole (DAPI; beyotime). Fluorescence images were captured using Eclipse Ci-L Fluorescence Microscope (Nikon, Tokyo, Japan).

The intact SGs were dissected and washed with 1 × PBS (Sangon Biotech, China), placed on a slide for fixation with 4% paraformaldehyde (Sangon Biotech, China), and permeabilized with Immunostaining Permeabilization Solution with Triton X-100 (Beyotime, China). The slides were then blocked with 5% fetal bovine serum (Beyotime, China) for 30 min, incubated with primary antibodies, all of which were commercially prepared by Zoonbio Biotechnology Co., Ltd, China, including fibH (#ZD17N03M5383), fibL (#ZD17N03M5385), P25 (#ZD17N03M5386), Ser1 (#ZD2018V002945), Ser2 (#ZD2018B002943), Ser3 (#ZD2018P002944), LOC101746180 (#ZD2021O013151), LOC101740197 (#ZD2021P013154), LOC101745308 (#ZD2021D013149), Btl (#ZD2021C013155) at a dilution ratio of 1:150 for 2 h, and then incubated with secondary antibody of anti-rat IgG (H + L) Alexa Fluor^® 555 Conjugate (#4417, CST, USA) at a dilution ratio of 1:150 for 1 h. Finally, the nuclei were stained with DAPI solution (Beyotime, China), and the slides were sealed with Antifade Mounting Medium (Beyotime, China) for observation. The fluorescence images were observed and analyzed using an inverted fluorescence microscope (Olympus, Japan). The antibody information is listed in Supplementary Table 2.