Anti fluorescence quenching sealing solution

Keratinase (Provided by Professor Shi Jinsong, School of Life Science and Health Engineering, Jiangnan University, School of Engineering, Jiangnan University), F12 medium, fetal bovine serum, 0.25% EDTA trypsin (Gibco, USA), streptomycin and penicillin (Thermo, USA), phosphate-buffered saline (PBS), CCK-8 kits, RNA rapid extraction kits (Yishan, China), 0.8 μm Transwell chambers, cell culture-grade DMSO, DAPI, Alexa Fluor 488 sheep anti-mouse, bromelain (Sigma, USA), FITC-dextran (MW4000) (MCE, USA), pentobarbital sodium, TNF-α and IL10 ELISA kit (Lianke Bio, China), fluorescence quantitative PCR kits, reverse transcription kits (TaKaRa, Japan), Arg-1 antibodies, iNOS antibodies, CD86 antibodies, CD206 antibodies, CD163 antibodies (Bio Legend, USA), H&E staining kits, Masson staining kits (Yeason, China), 4% paraformaldehyde, Tris–HCl, anti-fluorescence quenching sealing solution, immunohistochemical blocking solution (Beyotime, China), and primers (Sangon Biotech, China) were used.

After infection with bacteria containing the mCherry plasmid, the macrophages were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Subsequently, cells were washed with PBS and permeabilized with 0.1% Triton X-100 for 5 min. After washing with PBS, anti-fluorescence attenuation tablets (including DAPI) and anti-fluorescence quenching sealing solution (Beyotime, Shanghai, China) were dropped (10 μL at a ratio of 1:1) on the glass slide. The coverslips were mounted on slides, and cell images were acquired using a confocal laser scanning microscope (Zeiss LSM800) and analyzed with ZEN 2.3 (blue edition) and format designed using Adobe Illustrator CC 2018 software. The data were based on the clearest results preserved from five fields of three slides.

The rat liver tissue was fixed in 4% paraformaldehyde for 30 min, washed three times with a phosphate buffer solution, sealed with goat serum (Zsbio, Beijing, China) for 1 hour, and incubated overnight in a wet box with an anti-FAM134B antibody (Abcam, Cambridge, England) at 4°C, rewarmed in a wet box at 37°C for 30 min, and treated with fluorescein II at 37°C (Shanghai, China) incubated in the dark for 1 hour, stained with DAPI (Beyotime, Shanghai, China) for 5 min, sealed with an anti-fluorescence quenching sealing solution (Beyotime, Shanghai, China), and photographed with a fluorescence microscope (Motic, Shenzhen, China). Using Image-Pro Plus Version 6.0 (IPP) image analysis software, the integrated optical density (IOD) of each slice was calculated, and the IOD value was used to express the quantification index.