Histosec paraffin

For collection of the embryonic tissue, pregnant females were euthanized with CO₂ followed by cervical dislocation. The embryos were dissected in ice-cold PBS and immersed in freshly made 4% paraformaldehyde (PFA, Sigma-Aldrich) in PBS for fixation. For the adult brains, the mice were deeply anesthetized before pericardial perfusion using +37 °C PBS, followed by +37 °C 4% PFA in PBS. The adult brain tissue was post-fixed for 3–4 days at room temperature (RT). For paraffin embedding, the embryos were fixed at RT for 2–3 days, followed by dehydration and immersion in Histosec paraffin (Merck) using automated tissue processor (Leica TP1020). After embedding, 5 μm coronal sections were collected using a microtome (Leica HM360).
For frozen sections, the brains were dissected and post-fixed overnight in 4% PFA at +4 °C, and subsequently cryoprotected for 24–48 h in 30% sucrose after the pericardial perfusion. The brains were serially sectioned on a cryostat (NX70) at 12 μm.

On completion of in situ hybridization staining, embryos were fixed overnight in 4% PFA at 4 °C. Embryos were washed two times in PBST, then placed in plastic cryomoulds. PBST was removed and molten 1% agarose/PBS was added. Embryos were correctly orientated and left for 1 h to allow the agarose to set. The agarose block was removed from the mould and dehydrated serially in 70, 90, 95, 100% ethanol (Sigma-Aldrich) for 1 h each. The block was cleared in 50:50 ethanol:xylol (Sigma-Aldrich) then 100% xylol for 1 h each. To begin paraffin infiltration, the block was left in 50:50 xylol:HistosecParaffin (MerckMillipore) overnight at 60 °C. Hundred per cent paraffin was added and changed twice daily for 3 days. The block was then mounted on a holder and left at room temperature overnight before 6-μm sections were cut on a microtome. Sections were placed on a slide and mounted under a coverslip with Entellan (ThermoFisher).