Slot Blot mRNA Analysis
Five micrograms per sample of total RNA extracted from fresh-frozen brain, liver, kidney, heart, lung, spleen, and skeletal muscle was vacuum-filtered through 0.45-μm Optitran nitrocellulose. After several washes through the manifold with 10× SSC, blots were UV-cross-linked and probed with a radiolabeled ∼350-bp BglII–XhoI cDNA fragment of mo/huAPP695 cDNA. After hybridizing overnight at 65 °C in 1% BSA/1 mM EDTA/0.5 M sodium phosphate buffer (pH 7.2)/7% SDS [20], the blots were washed twice at 65 °C for 30 min each in 0.1% BSA/1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/5% SDS before two final 30-min washes at 65 °C with 1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/1% SDS. Blots were wrapped wet and exposed to phosphorscreens overnight at room temperature.