84 Chapter 4 (APPNL-G-F/NL-G-F) APP knock-in mice and 11 wild type mice. Mice were bred at Charles River Laboratories, UK and sampled at the Imperial College London, UK. Brain tissue samples were collected fresh from10- and28week-oldmice thatwereeuthanisedwith sodiumpentobarbital and exsanguinated. Animal procedures complied with national and institutional guidelines (UK Animals Scientific Procedures Act 1986) and adhered to 3R guidelines. Hippocampal areas were used as region of interest for characterization. TauP301S Male brain tissue was obtained from 10 homozygous P301S knock-in mice57-59 and 8 wildtype C57/Bl6-OLA mice (Envigo, UK) from the Centre for Clinical Brain Sciences, Edinburgh, United Kingdom. Brain tissue samples were collected from 8- and 20-week-old mice that were perfused with PBS and 4% paraformaldehyde, with tissues being post-fixed overnight before being cryopreserved in 30% sucrose and frozen embedded in tissue tec (Leica, UK). Sections were cut, 20mm, on a cryostat onto superfrost plus slides and stored in-80 freezer. Animal procedures complied with national and institutional guidelines (UK Animals Scientific Procedures Act 1986 & University of Edinburgh Animal Care Committees) and adhered to 3R guidelines. Hippocampal areas were used as region of interest for characterization. For all studies mice were housed 4-5 per standard cages in specific pathogen-free and controlled environmental conditions (temperature: 22±2°C; relative humidity: 55±10% and 12 h of light/dark). Food (standard pellets) and water were supplied ad libitum. Immunohistochemistry Paraffin sections were de-paraffinized by immersion in xylene for 5 min and rehydrated in descending concentrations of ethanol and fixed-frozen sections were dried overnight. After washing in PBS, endogenous peroxidase activity was blocked with 0.3 % H2O2 in PBS while for immunofluorescence sections were incubated in 0.1% glycine. Antigen retrieval was performed with citrate or TRIS/EDTA buffer, depending on the antibody, in a microwave for 3 min at 1000W and 10 min at 180W. Sections were cooled down to RT and incubated with primary antibodies (Table S6) diluted in antibody diluent (Sigma, U3510) overnight. Sections were washed with PBS and afterwards incubated with the appropriate secondary antibodies for 1 h at room temperature. HRP labelled antibodies were developed with diluted 3,3’-diaminobenzidine (DAB; 1:50, DAKO) for 10 min and counterstained with haematoxylin. Sections were immersed in ascending ethanol solutions and xylene for dehydration and mounted with Quick-D. For immunofluorescence, sections were incubated with Alexa Fluor®-labelled secondary antibodies. Autofluorescent background signal was reduced by incubating sections in Sudan black (0.1% in 70% EtOH) for 10 min. Nuclei were stained with 4,6-diami-dino-2-phenylindole (DAPI) and slides were mounted onto glass coverslips with FluoromountTM (Merck). Imaging mass cytometry Antibody conjugation was performed using the Maxpar X8 protocol (Fluidgm). 51 slides of paraffin-embedded tissue from the Medial Temporal Gyrus (MTG) and 48 slides of paraffinembedded tissue from the Somatosensory Cortex (SSC) underwent IMC staining and ablation. Each slide was within 5-10μm in thickness. The slides underwent routine dewaxing and rehydration before undergoing antigen retrieval, in a pH8 Ethylenediaminetetraacetic acid (EDTA) buffer. The slides were blocked in 10% normal horse serum (Vector Laboratories) before incubation with a conjugated-antibody cocktail (Table S6) at 4⁰C overnight. Slides
RkJQdWJsaXNoZXIy MTk4NDMw