Fitc goat anti mouse igg

NRK52e cells were uniformly inoculated at a density of 5 × 10⁴/mL in 96-well plates (PerkinElmer, MA, USA). The drug intervention was performed when the cell volume of each well was 80–90%. After the experiment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, then infiltrated with 0.1% Triton for 20 min. The treated cells were blocked with 5% goat serum at room temperature for 1 h and incubated with different primary antibodies at 4℃ overnight. Then incubated with secondary antibodies: Cy3 Goat Anti-Rabbit IgG (Abclonal, Wuhan, China) or FITC Goat Anti-Mouse IgG (Abclonal, Wuhan, China) for 1 h at room temperature. Finally, they were stained with DAPI for 10 min at room temperature after washing with PBS. High-content imaging system (PerkinElmer, MA, USA) was used to scan and obtain the images. The relative protein expression level was normalized using Harmony 4.8.

Immunofluorescence staining was performed according to our prior procedure [28 (link)]. Briefly, the cerebral tissues of rats were embedded in OCT and sliced by a cryosectioning machine (Leica, Germany) into coronal sections of 20 µm thickness. Cultured astrocytes were immobilized with paraformaldehyde (4%). Entire samples were blocked with goat serum albumin (10%) and overnight incubation of the samples at 4 °C was carried out using the primary antibodies shown in Table 2. Subsequently, the samples were incubated with FITC Goat Anti-Mouse IgG or Cy3 Goat Anti-Rabbit IgG (1:200, ABclonal, China) secondary antibodies, followed by the DAPI staining (Solarbio, China). A fluorescence microscope (Leica, Germany) was utilized for the imaging, and Image J was employed for the image analysis.