Nissl staining and Golgi staining

For Nissl staining, brains from perfused adult animals were post-fixed in 4% PFA overnight, dehydrated and paraffin-embedded. Sagittal and coronal sections were cut on a Microtome HM 355 at 10 µm thickness. For Nissl stainings in newborn mice, pups were decapitated at P0, brains dissected, drop fixed in 4% PFA, dehydrated in sucrose, embedded in O.C.T, and cut at 18 µm on a cryostat. Nissl staining with 1% Cresyl Violet solution (Cresyl Violet Acetate, Sigma, Cat.No C 5042) was performed upon clearance of paraffin slices with RotiHistol (Carl Roth) for 10 min and rehydration of sections (absolute EtOH to water: 96%, 90%, 70%, 50%, 30%, water, 3–5 min each), or 3 × 5 min washes in 1× PBS to remove the OCT. Nissl stainings of adult and P0/P1 brains were captured using an Olympus Slide scanner VS120 and analyzed using Fiji (v1.52n).

Golgi-Cox staining was performed according to protocol using the FD Rapid GolgiStain Kit^TM (FD Neurotechnologies). After three weeks of Golgi impregnation, brains were cut coronally (120 µm) using a Leica Vibratome (Leica VT 1200 s) and mounted onto 1% gelatin-coated slides. Slides were then dehydrated through graded ethanol steps, cleared with RotiHistol (Carl Roth), and mounted with DPX mounting medium on coverslips (#1.5). To quantitatively analyze pyramidal neurons in Golgi-stained slides, impregnated pyramidal cells (8–10 neurons per brain, n = 3 littermate brains per genotype) of layer 2/3 in the somatosensory cortex were selected and imaged with a Nikon Eclipse Ti2 using a ×40 magnification. For analysis, single pyramidal neurons were manually reconstructed using Imaris analysis software (version 9.3.1). The average filament area, filament length, and Sholl intersections were analyzed using the same software. Spine counting was performed using Fiji (v1.52n), spines that started from 100 µm distance of the apical dendrite were counted within a 100 µm segment.