Mice were sacrificed under anesthesia following NIH guidelines for the humane treatment of animals, and brains were removed. The right hemibrain was fixed by immersion in 4% paraformaldehyde in PBS pH 7.4 and serially sectioned at 40 μm with a Vibratome apparatus (Leica) for subsequent analysis. The left hemibrain was stored at −80 °C for biochemical analysis, and further processed for either quantitative real-time polymerase chain reaction (qPCR) or protein analysis.
Vibratome sections were immunolabeled overnight with antibodies against α-syn (Sigma, 1:250), Glial fibrillary acidic protein (GFAP) (Millipore, 1:500), Iba1 (Wako, 1:2000) or MAP2 (Millipore, 1:500), followed by incubation with species-appropriate secondary antibodies (Vector Laboratories). Sections were reacted with 3,3’-diaminobenzidine (Vector Laboratories) and imaged on an Olympus BX41 microscope. A minimum of 100 cells were counted per animal, and cell counts are expressed as the average number of positive cells per field (230 μm x 184 μm). Quantification of GFAP and Iba1 staining was performed by obtaining optical density measurements using the Image Quant 1.43 program (NIH) and corrected against background signal levels.
Vibratome sections were immunolabeled overnight with antibodies against α-syn (Sigma, 1:250), Glial fibrillary acidic protein (GFAP) (Millipore, 1:500), Iba1 (Wako, 1:2000) or MAP2 (Millipore, 1:500), followed by incubation with species-appropriate secondary antibodies (Vector Laboratories). Sections were reacted with 3,3’-diaminobenzidine (Vector Laboratories) and imaged on an Olympus BX41 microscope. A minimum of 100 cells were counted per animal, and cell counts are expressed as the average number of positive cells per field (230 μm x 184 μm). Quantification of GFAP and Iba1 staining was performed by obtaining optical density measurements using the Image Quant 1.43 program (NIH) and corrected against background signal levels.