Fluorescently labeled dextran

Bacteria were prepared from 3 mL overnight cultures, each derived from a single Escherichia coli colony, which were centrifuged and re-suspended in 500 μL of PBS. The 500 μL bacteria mix (~1.6 x 10⁶ cfu/μL) was supplemented with 1 μL of 5 mg/ml AlexaFluor 568 or AlexaFluor 488 conjugated dextran (10 kDa) from Invitrogen. Other reagents used for brain injections were: ultra-pure water supplemented with 0.05 mg/ml fluorescently labeled dextran (10 kDa) for the control water samples, and lipopolysaccharides (LPS) derived from 0111:B4 E. coli (L3024 Sigma) at 4.5 mg/ml was supplemented with 0.05 mg/ml fluorescently labeled dextran (10 kDa). For all injections, 1 nL of the reagent mix was injected into the right brain tectum of 4 dpf larvae, and the fluorescent dextran was used to validate the injection site. Larvae that were successfully injected were collected 6 hours post injection (hpi) and immediately processed for total RNA lysis. RNA extraction and subsequent steps for qPCR analyses are described above. All collected larvae derived from heterozygous intercrosses were genotyped after RNA extraction.