Streptavidin biotin peroxidase complex kit

Paraffin sections were used for immunohistochemical staining as described previously [23 (link)]. Briefly, sections were incubated with 3% hydrogen peroxidase to block endogenous peroxidase activity, with 5% bovine serum albumin to block nonspecific binding sites. Sections were incubated with polyclonal rabbit antihuman EP receptors (1:400 dilution for EP1, EP2, or EP3 and 1:200 for EP4, Cayman Chemicals, Michigan, USA) or polyclonal rabbit antihuman myeloperoxidase (MPO) (1:100 dilution, ZSGB-BIO, Beijing, China) overnight at 4 °C. The primary antibodies were detected with the StreptAvidin Biotin-peroxidase Complex kit (Boster Biotechnology, Wuhan, China). The immunoreaction was visualized using 3,3-diaminobenzidine-tetrahydrochloride, which causes brown staining of positive cells. Negative controls were performed by omitting primary antibodies and using non-immune sera of the same species.

Tissues from distinct developmental stages were fixed in 4 % paraformaldehyde (PFA) overnight at 4 °C. The fixed samples were processed in an alcohol gradient and xylene and then embedded in paraffin prior to being cut into 7-μm sections for IHC analysis. IHC was performed with the StreptAvidin-Biotin-peroxidase complex kit (Boster, Wuhan, China) for most tissues and the anti-rabbit IgG-HRP kit (Boster, Wuhan, China) for the liver and kidney according to the manufacturer’s protocols. The sections were incubated with primary antibody at 1:250 (CST or Genscript) in a humidified chamber overnight at 4 °C. The immunoreactive sites were visualized by incubation with freshly prepared 3,3′-diaminobenzidine-tetrachloride. The sections were then counterstained with hematoxylin and examined under a light microscope (Olympus, Narishige, Japan). In the same tissue, negative controls were used to check for non-specific development and were treated as described above, except that non-immune serum was used instead of primary antibody.