Zebrafish larvae at 4–10 dpf and 1 month old were mounted dorsal side up for brain injections or on their sides for intravenous injections in 1–3% low-melting agarose. A pneumatic microinjector (WPI) with a fine capillary glass pipette was used to inject 1 nL for 4–10 dpf or 2 nL for 1 month old of stimulus into the targeted site (either tectum for brain injections, or caudal vein plexus for intravenous injections). Lipopolysaccharides (LPS) derived from 0111:B4 E. coli (L3024 Sigma) at 5 ng/nL, ultra-pure water, or live Escherichia coli cells were supplemented with fluorescently labeled dextran (Invitrogen, 10,000 MW at 1:100 dilution of a 5 ng/nL stock) for visualization. E. coli cells were prepared for injections as previously described (Earley et al., 2018). Fluorescently tagged Alexa 594 conjugated LPS from 055:B5 E. coli (L23353 Sigma) at 5 ng/nL was used for brain microinjection at 1 nL per fish. All fish after microinjection are carefully monitored for normal health and behavior, and nearly all injected fish remain healthy and viable, showing no signs of overt change. These healthy post-injection fish are used for further experimentation and analysis.

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