Prolong gold antifade reagent p36930

Two rats were deeply anesthetized and transcardially perfused with saline. Whole brain tissues were removed and rapidly frozen on dry ice. The fresh-frozen tissue sections (20 μm thick) were mounted on positively charged microscopic glass slides (Fisher Scientific, Pittsburgh, PA). Both the vasopressin V1a and Gad1 specific RNA probes (Rn-Avpr1a, 402531 and Rn-Gad1, 316401) were designed and provided by Advanced Cell Diagnostics (Hayward, CA). All staining steps were performed following RNAscope protocols. Stained slides were coverslipped with fluorescent mounting medium (ProLong Gold Antifade Reagent P36930; Life Technologies) and examined with a confocal microscope (Leica TCS-SP5) at 63x magnification using the manufacturer-provided software.

The cellular localisation of DDX24 was studied using confocal microscopy. Paraffinised human brain tissue slides were deparaffinised and treated for antigen retrieval as described above. Samples were washed three times for five min in PBS-T and incubated with primary antibodies, anti-DDX24 (1:100) HPA 002554 with the immunogen ATP-dependent RNA helicase DDX24 recombinant protein epitope signature tag (PrEST) (Sigma-Aldrich, Saint Louis, MO, USA), anti-APP C-terminal C1/6.1 which have the epitope within amino acids 45–53 of APP (1:200), C1/6.1(BioLegend, San Diego, CA, USA), anti-amyloid precursor protein Y188 ab32136 (Abcam, Cambridge, UK) in PBS overnight (ON) at 4 °C. For negative control samples, the primary antibody was omitted. After washing three times for five min with PBS-T, a secondary incubation step was performed at RT 1 h with directly labelled secondary antibodies Alexa flour 647 and Alexa flour 555 at 1:500 in PBS (Sigma Aldrich, Saint Louis, MO, USA). After washing three times for five min, mounting was performed using ProLong gold anti-fade reagent P36930 (Life Technologies, Carlsbad, CA, USA), and slides were covered with coverslips with number 1.5. Samples were stored in darkness and at 4 °C when not in use.