Il 1β bs 0812r

Immunohistochemical analysis was performed using the 3,3N-Diaminobenzidine Tertrahydrochloride (DAB) horseradish peroxidase color development kit (P0203, Beyotime) as previously described [22 (link)]. The sections were incubated with primary antibodies targeting tumor necrosis factor-α (TNF-α) (ab6671, Abcam, Cambridge, U.K.), interleukin-6 (IL-6) (SC-1265, SantaCruz, Dallas, TX, USA), Interleukin-1beta (IL-1β) (bs-0812R, Bioss, Woburn, MA, USA). After being mounted with neutral resin, the sections were observed under a light microscope (Olympus, Japan) and analyzed using the ImageJ software.

The sections were incubated with primary antibodies (IL-1β (bs-0812R, Bioss, Beijing, China; 1:200); TNFa (AF7014, Affinity, Ohio; 1:200); iNOS (AF0199, Affinity, Ohio; 1:200)) at 4℃overnight. HRP-AffiniPure Goat Anti-Rabbit IgG (H + L) (BM3894, Boster, Wuhan, China; 1:1000) was incubated at 37℃for 30 min. The staining was displayed using Diaminobenzidine. Cells with only blue nuclei are negative, and cells with brownish yellow or tan granules in the cytoplasm are positive cells. The staining intensity was scored as the following: 0 point for no staining; 1 point for weak positive (light yellow staining); 2 point for moderated positive (brownish yellow); 3 point for strong positive. The positive cells in three randomly selected field were scored as the following: 0 point for negative; 1 point for less than 10%; 2 point for 11–50%; 3 point for 51–75%; 4 point for more than 75%.