Polyclonal sheep anti rat albumin

The Western blotting analysis was performed as reported previously [38 (link)]. Briefly, periventricular or cortical tissues were collected and sonicated in Western blotting sample buffer. Protein concentration was detected by the Bio-Rad protein assay kit, and 40 μg protein samples were loaded and then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before transfer to a Hybond-C pure nitrocellulose membrane (Amersham, St. Louis, MO, USA). Membranes were probed with the following primary antibodies: polyclonal rabbit anti-HO-1 (1:2000 dilution, Enzo, Farmingdale, NY, USA) and polyclonal sheep anti-rat albumin (1:10,000 dilution, Bethyl Laboratories, Montgomery, TX, USA). Antigen–antibody complexes were visualized with the ECL technique. Image analysis was done with the ImageJ software by a masked investigator.

The brains were immersed in 4% paraformaldehyde before transfer to a 30% sucrose in 0.1 M cold phosphate-buffered saline (pH 7.4) solution for 2–3 days at 4 °C to avoid crystal formation. The brains were embedded before sectioning coronally to 18 μm on a cryostat. Immunohistochemistry was performed with the avidin–biotin complex technique. Sections were blocked by 1:10 goat or horse serum (Vector Laboratories,Burlingame, CA, USA) at room temperature for 30 min and then incubated at 4 °C overnight with the primary antibody. The primary antibodies were polyclonal rabbit anti-HO-1 (1:400 dilution, Enzo, Farmingdale, NY, USA), monoclonal mouse anti-rat CD163 (1:50 dilution, Bio-Rad, Hercules, CA, USA), and polyclonal sheep anti-rat albumin (1:4000 dilution, Bethyl Laboratories, Montgomery, TX, USA). The secondary antibodies included goat anti-rabbit IgG (1:500 dilution, Bio-Rad, Hercules, CA, USA), horse anti-mouse IgG (1:500 dilution, Bio-Rad, Hercules, CA, USA), and horse anti-sheep IgG (1:500 dilution, Bio-Rad, Hercules, CA, USA). Negative controls were obtained by the omission of the primary antibody.