The method has been shown to be highly reliable and robust for RNA samples isolated from Formalin Fixed Paraffin Embedded (FFPE) tissue samples such as mouse brain samples which are available from a large number of previous experiments in our laboratory as archived probes (Reis et al., 2011; Chen et al., 2016). a total of twelve times with DNA A42 trimer using gene gun delivery over Rabbit polyclonal to ADAMTS1 a time frame of 24 months. Control mice received A1C42 peptide immunizations (100 g peptide plus QuilA adjuvant, i.p.injections) or were left untreated. The DNA plasmids and immunization with the gene gun had been described previously in detail (Qu et al., 2010; Rosenberg et al., 2018), Immunizations were started in four months old mice in groups of ten to fifteen mice (3xTg-AD and B6129/SvJ wild-type controls) with three initial immunizations in biweekly intervals via i.p. injections of A1C42 peptide (100 g peptide/immunization) with QuilA as adjuvant. The immunizations were boosted in six week intervals until the mice were 24 months old. Control mice received Orphenadrine citrate no treatment (na?ve controls). No immunizations were performed during or close to the behavior studies to avoid behavioral variances due to stress. Blood samples were collected at different time points throughout the study 10 days following the respective immunization time points. 2.3. Nesting behavior Mice were placed individually in fresh cages, in which 6 g of shredded paper were dispersed throughout the cage. Pictures were taken after 24 h and scored on a scale of 1C5. A score of 1 1 represented no nest constructed and 5 represented a fully formed nest, with all of the paper in the cage being gathered tightly in one area (Suppl. Fig. 1). 2.4. ELISAs Antibody concentrations in plasma were measured with standard ELISA assays and determined as g anti-A42 IgG/ml plasma. Plates were coated with A1C42 peptide (rPeptide) at concentrations of 2 Orphenadrine citrate g/ml diluted in coating buffer. As standard antibody the mouse monoclonal anti-A17C24 IgG antibody 4G8 (Biolegend, San Diego, CA) was used. Antibody binding to A42 peptide in the plates were measured with HRP conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch laboratories, West Grove, PA), TMB detection and measurement of optical density (OD) with an ELISA plate reader. 2.5. IFN and IL-17 FluoroSpots FluoroSpot assays to determine frequencies of cytokine secreting cells were performed according to standard procedures and as previously described using commercial available antibody sets for mouse IFN, IL-17, and IL-4 (Maptech, San Diego, CA, USA) (Lambracht-Washington et al., 2011; Lambracht-Washington and Rosenberg, 2015). 2.5 105 splenocytes were added per well and were cultured in medium only, or re-stimulated with A42 peptides (10 g/ml), and incubated at 37 C in a 5% CO2 humidified incubator for 48 h. For maximal T cell stimulation Orphenadrine citrate an anti-CD3 antibody (clone 145C2C11, eBioscience, Tonbo) was used at 0.5 g/ml in the cell cultures. Spots were counted by a FluoroSpot plate reader service (Zellnet consulting, Fort Lee, NJ). 2.6. Memory T cell FluoroSPOT Splenocytes from immunized and non-immunized mice were cultured for 10 days with and without A42 antigen stimulation and IL-2 supplementation (10 ng/ml) at Day 3 and Day 7 of cell culture. On day 10, cells were harvested and plated onto pre-coated IFN FluoroSPOT plates and re-stimulated overnight with full-length A1C42 peptide, and the shorter peptide A10C26 (106 cells/well). IFN secreting cells (spots) were detected with a biotinylated anti-IFN antibody and visualized with AF550. Plates were read by a commercial plate reader service (Zellnet). 2.7. Immunohistochemistry of mouse brains Mice were overdosed with avertin and perfused transcardially with cold PBS. Brains were extracted from the skulls and kept.