Triton x 100

After in vitro infection for 24 h, cells were fixed with 4% paraformaldehyde for 1 h, permeabilized with 0.1% Triton X-100 (Biosharp) for 30 min, and blocked with 1% FBS for 1 h. H. pylori, lipid rafts, cytoskeleton, and nucleus were stained with anti–H. pylori antibody (Biosharp), fluorescein isothiocyanate-labeled cholera toxin subunit B (FITC-CTX-B) (Absin), rhodamine-labeled phalloidin (Biosharp), and 4, 6-diamidino-2-phenylindole (DAPI) (Biosharp), respectively, and fluorescence signals were analyzed using a confocal laser scanning microscope (Carl Zeiss).

Free-floating sections were washed in PBS, incubated for 1 h in blocking solution (1% BSA (Macklin; China) at 37 °C and 0.3% Triton X-100 (Biosharp, China) in PBS and incubated overnight at 4 °C with primary antibodies (see below for a list of antibodies) in blocking solution. For c-Fos staining, slices were incubated with the primary antibody for one night at 4 °C. Sections were then washed with PBS and incubated for 2 h at 37 °C with secondary antibodies (see below for a full list of antibodies) in 0.3% Triton X-100 in PBS. Finally, sections were washed in PBS, incubated with 4,6-diamidino-2-phenylindole (DAPI:1 µg/5 ml, Sigma, America) and mounted on slides with mounting medium (50% Glycerol anhydrous in PBS, Biofroxx, Germany).

Mice were euthanized and perfused with saline and then ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS) for 12–24 h, then the brains were placed in 30% sucrose solution for 24 h and cut into 20 μm slices by machine of frozen slicer (Leica CM1950). For immunofluorescence, sections are fixed in 4% PFA for 10 min and rinsed in PBS for 10 min each time for three times. Subsequently, sections are permeabilized in 5% donkey serum (Jackson Immuno Research, USA) containing 0.3% Triton X-100 (Biosharp, South Korea) at room temperature and incubated at 4 °C overnight. TH primary antibody (1:1000, Abcam, UK) and anti-α-synuclein filament antibody [MJFR-14-6-4-2]-Conformation-Specific (1:5000 Abcam, UK) were diluted in 0.01 M PBST. The sections were incubated with a secondary antibody (1:500 Invitrogen, USA) for fluorescence staining at room temperature for 2 h. The sections were then mounted onto glycerin-coated slides and cover slipped. The images were obtained by a confocal microscope (Olympus VS120, Japanese) and quantifications were performed by the software OlyVIA\xvViewer and ImageJ.

Cells were seeded into 6‐well plates in which 14‐mm cell climbing slices had settled. Cells were grown to a suitable density, washed with ice‐cold PBS, fixed with 4% paraformaldehyde, washed with PBS three times, permeabilized with Triton X‐100 (Biosharp, CHN), and blocked with goat serum at room temperature in sequence. The slices were incubated with primary antibodies for 2 h at room temperature and then with Cy3‐labeled or FITC‐labeled secondary antibodies for 1 h. DAPI was used to stain the cell nucleus. A fluorescence microscope was used to capture the images.

HCAECs were fixed with 4% paraformaldehyde (Biosharp Biotechnology, China, Cat No: BL539A) for 30 min and permeabilized with 0.1% Triton X-100 (Biosharp, China, Cat No: 1805132) for 20 min. After washing with PBS, 5% bovine serum albumin (BSA) in PBS was added and incubated at room temperature for 2 h. Cells were then incubated with anti-LC3 (1 : 500, CST, USA, Cat No: 3868S) and p62 (1 : 200, CST, USA, Cat No: 88588S) antibodies overnight. The second fluorescence was incubated in the dark for 1 h. The nuclei were stained with DAPI for 5 min, and images were evaluated by confocal microscopy (OLYMPUS, Japan).

Mtdh and nephrin (podocyte marker) antibodies were used to investigate the location and expression of Mtdh in frozen renal tissue samples obtained from db/db and db/m mice. The slides were permeabilized with 0.05% Triton X-100 (Biosharp, Anhui, China) in PBS for 10 min and blocked with 5% goat serum mixed with 2.5% bovine serum albumin for 2 h. Afterward, these sections were incubated with anti-Mtdh antibody (1:100; Abcam) together with anti-nephrin antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. Anti-Mtdh and anti-nephrin primary antibodies were detected using Alexa Fluor 546 donkey anti-rabbit and Alexa Fluor 488 goat anti-rabbit (1:1000; Invitrogen) secondary antibodies, respectively, and the samples were incubated with them for 6 h at 4 °C. Cell nuclei were stained with DAPI for 10 min before the observation of the samples under a light microscope (Nikon Corporation).