*4.7. Analysis of Microglia*

Slides were washed and then blocked using normal donkey serum followed by rabbit anti-Iba1 polyclonal antibody (1:500; Wako; 019-19741) overnight at 4 ◦C. This Iba1 antibody recognizes the carboxy-terminal of the Iba1 protein and is specific to microglia/macrophages. Slides were washed and incubated for 2 h in donkey anti-rabbit TRITC (JacksonImmuno, West Grove, PA, USA; 131591) at room temperature. After washing, slides were mounted using Vectashield Mounting Media with DAPI and placed on coverslips. Images were captured using confocal microscopy with a 40X objective. Iba1+ cells were counted in the sub-ventricular zones (SVZ) associated with the third ventricle. A total of 2 sections per fetus were used for microglial assessment. Images of the third ventricle's open and closed regions were captured from each fetal brain. Microglia totals from 1–2 pups were averaged per dam. Counting was done in a blind fashion. At E19, microglia have a di fferent morphology from those in the adult brain; and amoeboid and primitive ramified microglia are most commonly observed [31]. We therefore counted the number of amoeboid and primitive ramified microglia in the SVZ. Image analysis was done using ImageJ (version 1.51j8; NIH). Cortical images were captured using a 10X objective, and the thickness of the cortical plate was analyzed using NIS Elements' Analysis software (Nikon Instruments Inc., Melville, NY, USA; version 5.10.01). A total of 6 measurements per pup brain were obtained within the cortical plate and averaged per pup, and further averaged per dam.
