*4.5. Micro-Bleed Detection*

Slides were washed and stained using hematoxylin and eosin following the manufacturer's directions. Slides were then cover-slipped and imaged using light microscopy. Micro-bleeds were identified as red cells within the parenchyma and outside of the blood vessel lumen or within the ventricles. Micro-bleeds were counted by an investigator blinded to the groups (ABG) in sections from the anterior and posterior part of the brain (Figure 2A). The number of micro-bleeds from two slices per region was averaged per fetus and further averaged per dam.

### *4.6. Fetal Brain Multiplex Array*

To determine the cerebral inflammatory profile of exposed fetuses, a separate group of brains (*n* = 1 pup per dam) were homogenized in RIPA bu ffer, and protein concentration was measured using the BCA kit. Equal volumes (25 μL) of sample were loaded into 96 well-plates and incubated with magnetic beads, pre-mixed to detect 27 cytokine/chemokines (Rat multiplex kit, Millipore Sigma, Burlington, MA, USA). Samples were run alongside standards and kit controls in duplicate. The observed concentration was calculated using the standard curve generated and normalized to the protein concentration of the sample. Cytokine/chemokine concentration is, therefore, presented as pg/mg protein.
