Figure 5

Flow diagram of the optimized protocol with possible stop-points (red boxes). All steps were performed in an RNase -free environment. Fresh-frozen whole heads were stored at -80°C for up to several months. Heads were cryosectioned at 20 μm, and four to six sections were mounted on a membrane-coated PEN slide. Sections were fixed in 95% EtOH, rehydrated with 75% and 70% EtOH, stained with 1% cresyl violet in 70% EtOH and dehydrated with 70%, 75%, 95%, 100%, 100% EtOH, 30 sec each. Slides were stored at -80°C up to several weeks in a box with desiccant beads. Four cell groups from the same section were microdissected, harvested in individual tubes with RNA stabilization solution and stored at -80°C. Stabilization solution was replaced with lysis buffer and cell lysates from four brains were pooled before RNA extraction.