Envision plus system hrp labeled polymer

After the tests of abnormal behavior, mice were anesthetized and transcardially perfused with ice-cold PBS. Their brains were then removed and processed for optical microscopy or confocal fluorescence microscopy as previously reported [6 ]. For optical microscopy, a rabbit polyclonal antibody against ionized calcium-binding adaptor molecule 1 (IBA1; Wako, Osaka, Japan) was used as the primary antibody. The secondary antibody was EnVision-plus system HRP-labeled polymer (anti-rabbit; Dako, Glostrup, Denmark). Immunoreactivity was developed and visualized by use of DAB substrate (SK-4100; Vector Laboratories, Burlingame, CA, USA) and quantified by using ImageJ software (NIH, Bethesda, MD, USA). For confocal fluorescence microscopy, the primary antibodies used were goat anti-COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich, St. Louis, MO, USA); and the secondary antibodies were Alexa Fluor 488-labeled donkey anti-goat IgG (H + L) (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568-labeled goat anti-mouse IgG (H + L), respectively. The mounting medium used was VECTASHIELD (Vector Laboratories, Burlingame, CA, USA). Images of the hippocampus were captured with a confocal fluorescence microscopy system (LSM510; Zeiss, Oberkochen, Germany).

On day 22, mice were transcardially perfused with ice-cold PBS, and brains were removed and postfixed with 4% paraformaldehyde. 30 μm-thick coronal sections of frozen brains were cut using a cryostat (CM3050S; Leica Microsystems, Heidelberger, Germany). Brain sections were immunostained using a rabbit polyclonal antibody against IBA1 (Wako, Osaka, Japan) to stain microglia/macrophages, a rabbit polyclonal antibody against TH (Cell signaling, Woburn, MA, USA) to stain dopaminergic neurons, and mouse monoclonal antibody against GAD67 (Abcam, Cambridge, UK) to stain GABAergic neurons. The secondary antibody used was an EnVision-plus system HRP-labeled polymer (anti-rabbit or anti-mouse; Dako, Glostrup, Denmark). Immunoreactivity was developed and visualized by DAB substrate (SK-4100; Vector Laboratories, Burlingame, CA, USA). Regarding the quantification of IBA1-positive signals, ImageJ software (NIH, Bethesda, MD, USA) was used as previously described [10 (link)], and TH and GAD67-positive signals were manually counted.