Immunohistochemical Analysis of NAGLU+/+ and NAGLU-/- Mouse Brains

NAGLU +/+and NAGLU^−/− mice were anesthetized and transcardially perfused with saline solution containing 0.01 mL heparin, followed by 4% paraformaldehyde in 0.1 mol/L PBS saline solution. Brains were processed as previously described.⁷⁵ (link) Briefly, brains were rapidly removed on ice and postfixed overnight at + 4°C and cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB) with sodium azide 0.02% for 24 h at 4°C. Brains were sectioned frozen on a sliding cryostat at 40 μm thickness, in rostrum-caudal direction. Afterward, free floating serial sections were incubated with PBS Triton X-0.3% and blocking solution (0.5% milk, 10% FBS, 1% BSA) for 1 h and 30 min. The sections were incubated overnight at + 4°C with the primary antibody anti-COX1. The sections were then incubated with the corresponding florescent-labeled secondary antibodies, Alexa 488/Alexa 594 conjugated antimouse/antirabbit IgG. Nuclei were counterstained with Hoechst. Images were observed using a Zeiss LSM700 META/laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Single images were taken with a resolution of 1024 × 1024.⁷⁶ (link)