Zebrafish (Danio rerio) were maintained at 28°C in a 13 h light and 11 h dark cycle. Embryos were collected by natural spawning and raised at 28.5°C in E3 solution according to standard protocols (Westerfield, 2000 ). Experimental protocols were approved by Macquarie University Animal Ethics Committee (Zebrafish models of neural disorders; protocol no. 2012/050).
The behavior of “activated” microglia was studied between 48 hours post fertilization (hpf) and 5 days post fertilization (dpf). In order to allow high-resolution confocal live-imaging of individual neuron-microglia interactions, we utilized the following previously characterized zebrafish lines: Tg(mpeg1:GAL4,UAS:mCherry) (gl22Tg), referred to as mpeg1:mCherry in the text (Ellett et al., 2011 (link)); rwTg(isl1:GFP), referred to as islet1:GFP in the text (Higashijima et al., 2000 (link)); Tg(met:GAL4,UAS:EGFP) (ed6), referred to as cmet:GFP in the text (Hall et al., 2007 (link)); s1020tEt(-0.6hsp70l:GAL4-VP16) (s1020t) and Tg(UAS:Kaede), referred to as s1020t:Kaede in the text (Scott et al., 2007 (link)). Expression constructs and novel transgenic lines were generated using the Tol2 kit (Kwan et al., 2007 (link)). Tg(mnx1:mKOFP2-CAAX) (mq7Tg) (Flanagan-Steet et al., 2005 (link); Arkhipova et al., 2012 (link); Acosta et al., 2014 (link)), referred to as mnx1:mKO2 in the text was generated using recombined p5E-mnx1 (-6 to -2869bp mnx1, Arkhipova et al., 2012 (link)), pME-mKO2caax (synthesised by GeneArt), p3E-pA (Kwan et al., 2007 (link)) and pDest-Tol2-pA2-acrys-EGFP (Berger and Currie, 2013 (link)). Tg(-3.5ubb:secAnnexinV-mVenus) (mq8Tg), referred to as ubiq:secAnnexinV-mVenus in the text was generated using recombined p5E-ubb (Mosimann et al., 2011 (link)), pMEsecAnnexinA5-NS (Addgene ID 67718), p3E-mVenus (ID 67719) and pDEST-Tol2-pA2 (Kwan et al., 2007 (link)). pME-secAnnexinA5-NS was based on the initial design of Van Ham et al. (2010 (link), 2012 (link)). It incorporates the human ANXA5 fused to a mammalian codon optimized consensus secretion signal. The ubiq:secAnnexinV-mVenus fish line expresses fluorescent AnnexinV ubiquitously throughout the zebrafish and allows the detection of any cell that expresses phosphatidylserine (PS) on the outer leaflet of the plasma membrane. PS is normally constrained to the inner leaflet of the plasma membrane and gets exposed to the outer leaflet in various conditions, including oxidative stress and apoptosis (Kuan et al., 2000 (link); Valencia and Morán, 2001 (link)).
The behavior of “activated” microglia was studied between 48 hours post fertilization (hpf) and 5 days post fertilization (dpf). In order to allow high-resolution confocal live-imaging of individual neuron-microglia interactions, we utilized the following previously characterized zebrafish lines: Tg(mpeg1:GAL4,UAS:mCherry) (gl22Tg), referred to as mpeg1:mCherry in the text (Ellett et al., 2011 (link)); rwTg(isl1:GFP), referred to as islet1:GFP in the text (Higashijima et al., 2000 (link)); Tg(met:GAL4,UAS:EGFP) (ed6), referred to as cmet:GFP in the text (Hall et al., 2007 (link)); s1020tEt(-0.6hsp70l:GAL4-VP16) (s1020t) and Tg(UAS:Kaede), referred to as s1020t:Kaede in the text (Scott et al., 2007 (link)). Expression constructs and novel transgenic lines were generated using the Tol2 kit (Kwan et al., 2007 (link)). Tg(mnx1:mKOFP2-CAAX) (mq7Tg) (Flanagan-Steet et al., 2005 (link); Arkhipova et al., 2012 (link); Acosta et al., 2014 (link)), referred to as mnx1:mKO2 in the text was generated using recombined p5E-mnx1 (-6 to -2869bp mnx1, Arkhipova et al., 2012 (link)), pME-mKO2caax (synthesised by GeneArt), p3E-pA (Kwan et al., 2007 (link)) and pDest-Tol2-pA2-acrys-EGFP (Berger and Currie, 2013 (link)). Tg(-3.5ubb:secAnnexinV-mVenus) (mq8Tg), referred to as ubiq:secAnnexinV-mVenus in the text was generated using recombined p5E-ubb (Mosimann et al., 2011 (link)), pMEsecAnnexinA5-NS (Addgene ID 67718), p3E-mVenus (ID 67719) and pDEST-Tol2-pA2 (Kwan et al., 2007 (link)). pME-secAnnexinA5-NS was based on the initial design of Van Ham et al. (2010 (link), 2012 (link)). It incorporates the human ANXA5 fused to a mammalian codon optimized consensus secretion signal. The ubiq:secAnnexinV-mVenus fish line expresses fluorescent AnnexinV ubiquitously throughout the zebrafish and allows the detection of any cell that expresses phosphatidylserine (PS) on the outer leaflet of the plasma membrane. PS is normally constrained to the inner leaflet of the plasma membrane and gets exposed to the outer leaflet in various conditions, including oxidative stress and apoptosis (Kuan et al., 2000 (link); Valencia and Morán, 2001 (link)).
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