Anti rabbit igg alexa flour 488

Paraffin sections were deparaffinized sequentially in xylene and different concentrations of ethanol. The sections were immersed in citrate and subject to moderate to high temperature repair. After cooling, they were incubated with 10% goat serum for 30 min at 37°C. For immunohistochemical staining, an anti‐TH (1:500, Proteintech) antibody was added after discarding the goat serum and incubated overnight at 4°C. After washing by PBS, biotinylated secondary antibody working solution was dropped and reacted at 37°C for 20 min. After washing with PBS again, the working solution of horseradish conjugated streptavidin was dropped and reacted at 37°C for 20 min. The sections were stained with Diaminobenzidine (DAB) and then photographed. For immunofluorescence staining, the following primary antibodies were incubated at 4°C overnight: anti‐TH (1:200; Proteintech), IBA‐1 (1:250; Abcam), and NLRP3 (1:200; Proteintech) antibodies, and then washed with PBS and incubated at 37°C for 60 min in the dark with secondary antibodies: anti‐rabbit IgG Alexa Flour 488 (1:500; Abcam) or anti‐rabbit IgG Alexa Flour 594 (1:500; Abcam) antibodies. After PBS rinsing, the slides were mounted with glycerol. All the results were analyzed and processed with ImageJ software after photography.

Rats were perfused intracardially with PBS and subsequently fixed with 4% paraformaldehyde. After tissue was dehydrated and embedded in paraffin, a total of 5-μm section was cut and collected. Antigens were repaired via high temperature and pressure antigen repair using citrate buffer (pH=6). Tissue was cooled to room temperature and incubated in goat serum for 30 min, and incubated with the following primary antibodies at 4 overnight: rabbit anti-TH (1:500; Abcam), rabbit anti-IBA-1 (1:300; Abcam), rabbit anti-GFAP (1:500; Proteintech), rabbit anti-ΔFOS B (1:100; HuaAn Biotechology). For immunohistochemical staining, slides were incubated with biotinylated secondary antibody at 37°C for 15 min and visualized with avidin horseradish enzyme at 37°C for 20 min. Slices were visualized through DAB kit. For immunofluorescence staining, the second antibodies were anti-rabbit IgG Alexa Flour 488 (1:800; Abcam). Quantification of TH-positive neurons was performed through visually counting the number of TH-positive neuronal cell bodies blindly by two investigators. The images were recorded with a charge-coupled device camera and operated with the MetaMorph software. The results were obtained from the average. The mean value for the number of TH-positive neurons in substantia nigra was then deduced by averaging the counts of six sections for each rat.