Aperio at2 digital pathology scanner

Brains were quickly isolated and fixed in 10% neutral‐buffered formalin (NBF) for 24-h, followed by ethanol gradient dehydration and infiltration with melted paraffin in an automated processor. An array of 4 µm coronal sections of whole-brain was prepared, to collect different regions of the cortex, and RNAscope in situ hybridization was performed according to the manufacturer’s reference guide, using the single‐plex RNAscope® assay kit-BROWN (Advanced Cell Diagnostics) on a Leica Biosystems BOND RX platform, as described previously^{64 (link)}. Brain slices were baked and deparaffinized on the instrument, followed by target retrieval and protease treatment. Hybridization was performed by using probe Mt1 (ACD, 547711), Tsc22d3 (ACD, 448341), Gjb6 (ACD, 458811) and Atp1b2 (ACD, 417131) followed by amplification as these genes were among our top hits (based on FDR and fold changes), DAB chromogenic detection and counterstain with haematoxylin. Imaging of sections was carried out on a Leica Biosystems Aperio AT2 Digital Pathology Scanner and analysis performed using Visiopharm software.

Hippocampal sections from paraffin embedded postmortem AD brain tissue were cut at a thickness of 5 microns and allowed to dry overnight in a 60°C oven. Following de-paraffinization and rehydration, target retrieval was performed by steaming the sections for 30 min in deionized water. Immunostaining was performed with a Dako Autostainer using Envision Plus kit (Agilent, K4011). Endogenous peroxidase was blocked for 5 min with 0.03% hydrogen peroxide. Sections were then treated with 5% normal goat serum (Invitrogen, 16,210,072) for 20 min. Subsequently, sections were incubated for 45 min at room temperature in different dilutions of primary Abs against p-S65-Ub (Ab A-D). After incubation with primary Ab, sections were incubated in Envision-Plus rabbit- or mouse-labeled polymer HRP (Agilent, K4011) for 30 min at room temperature. Peroxidase labeling was visualized with the chromogen solution 3, 3ʹ-diaminobenzidine. The sections were then counterstained with Lerner 1-hematoxylin (Fisher Scientific, CS400-1D) and cover slipped with Cytoseal mounting medium (Thermo Scientific, 8310). After drying, all sections were scanned with an Aperio AT2 digital pathology scanner (Leica Biosystems, Wetzlar, Germany).