Tritc conjugated anti rabbit igg

After 3 days of DBI, the rat brains were perfused with cold PBS and quickly embedded in OCT medium (Sakura, USA), after which 8‐μm‐thick sections were prepared. The sections were subsequently fixed in cold acetone at 4°C for 5 min, blocked with 3% BSA at room temperature for 30 min, and then incubated with the following primary antibodies at 4°C overnight: Iba‐1 (1:500, Abcam, catalog number: ab5076), AQP4 (1:500, Cell Signaling Technology, catalog number: 59678T), CD31 (1:500, R&D Systems, catalog number: AF3628), and GFAP (1:500, Abcam, catalog number: ab48004). Then, the sections were incubated with Tritc‐conjugated anti‐rabbit IgG (1:100; Zsgb‐bio) at room temperature for 1 h in the dark. DAPI was used as a counterstain for 5 min. The negative controls were treated with PBS. Finally, the slices were visualized under a fluorescence microscope at ×200 magnification, and the positive cells in matched sections were counted using ImageJ software (version 1.48v, NIH).
Brain cell apoptosis was evaluated using a fluorometric terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) system (Promega, Madison, WI). Negative controls were prepared with the same procedures but without the primary antibody. The positive areas of polarized light microscopy with second harmonic generation (PSR) were quantified using ImageJ software.

Primary hepatocytes and NCTC1469 cells were fixed with acetone and blocked with 5% FBS at room temperature for 30 min. Rabbit anti-mouse Igκ (Proteintech, Rosemont, IL, USA) was added at 4 °C overnight. After washing with PBS, FITC-conjugated anti-rabbit (ZSGB-BIO, Beijing, China) and tetramethylrhodamine–isothiocyanate (TRITC)-conjugated anti-rabbit IgG (ZSGB-BIO, Beijing, China) were added at room temperature for 1 h. Sections were visualized and imaged with a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan) or a confocal laser-scanning microscope (LSM 800; Carl Zeiss, Oberkochen, Germany). Quantification of fluorescence intensity for Igκ or cleaved caspase-3 was performed using ImageJ version 1.52.