Frozen brains (n = 2 males per age; aged 6, 18 and 21 months; C57BL/6J strain) were embedded in OCT for cryosectioning at 16 micron thickness (app. Bregma -1.655mm; Allen brain reference atlas coronal section 71). Reactions were carried out with the Visum Spatial Gene Expression (GEX) and Tissue Optimization (TO) Slide & Reagent Kits according to the manufacturer's protocol with recommended reagents (10X Genomics, 1000193 and 1000184). Sections were placed on designated capture areas of slides for TO and GEX and stored at -80°C until further processing. GEX slides were fixed with methanol and stained with hematoxylin and eosin (HE) for visualization of tissue morphology on a AxioImager Widefield Fluorescence Microscope (Zeiss) at 10-fold magnification. GEX slides were incubated with permeabilization enzyme for 20 minutes followed by incubation with RT and second strand synthesis. cDNA was then transferred into tubes and amplified for 15 cycles using a Thermal Cycler (BioRad). Library construction steps were performed according to the manufacturer's protocol and included cDNA fragmentation, end repair and A-tailing, adaptor ligation, and sample indexing and amplification. Quality control of the constructed library was conducted via Bioanalyzer (Agilent). 10X Visium for flash frozen tissue, v3 chemistry