Tracing VDA-derived HSPC Colonization in CHT

Fluorescent tracing of VDA-derived HSPCs colonizing the CHT was done using the tg(kdrl:Dendra2) line as described before [32 (link), 33 (link)] with a Leica SP5 confocal microscope with a ×20 dry objective. At 28 hpf an area of ~40 × 750 nm around the VDA, parallel to the yolk sac extension was photoconverted. The 405 nm UV laser intensity and exposure time were optimized for strong Dendra2-conversion without cell damage. After photoconversion embryos were transferred to E3 medium and at 50–60 hpf their CHT areas were imaged on a Leica SPE Live confocal microscope using a ×20 dry objective. To exclude bleed-through of Dendra-green, red channel detection was set stringently (630–680 nm).

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Blokzijl-Franke S., Ponsioen B., Schulte-Merker S., Herbomel P., Kissa K., Choorapoikayil S, & den Hertog J. (2021). Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells. Oncogene, 40(15), 2741-2755.

Publication 2021

SP5 confocal microscope SPE Live confocal microscope

Cell Confocal microscope Embryos Spe a Yolk sac

Corresponding Organization : Leiden University

Other organizations : Utrecht University, Institut Pasteur, Centre National de la Recherche Scientifique, Inserm, Université de Montpellier

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Variable analysis

independent variables

Photoconversion of an area of ~40 x 750 nm around the VDA, parallel to the yolk sac extension at 28 hpf using a 405 nm UV laser

dependent variables

Fluorescent tracing of VDA-derived HSPCs colonizing the CHT

control variables

Leica SP5 confocal microscope with a x20 dry objective used for photoconversion
Leica SPE Live confocal microscope with a x20 dry objective used for imaging at 50-60 hpf
Stringent red channel detection (630-680 nm) to exclude bleed-through of Dendra-green

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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