Microinjections were performed on larvae at 72 hpf using a Narishige IM-300 Microinjector and pulled thin wall glass capillaries (Harvard Apparatus), administered under anaesthesia by intravenous microinjection through the cardiac sinus venosus (SV) that drains the common cardinal vein (CCV). An injection volume of 1 nL was used for all intravenous injections to minimise disruption to blood volume.
For propidium iodide intravital staining, 1nL 100μg/ml propidium iodide in DPBS was injected immediately following injury at 0.5 hpi. Larvae were then immediately imaged by heartbeat-synchronised light-sheet microscopy at 1 hpi. Injection of recombinant zfIFN-γ-rel (IFN-1.1) (Kingfisher Bioscience) was administered as a single 1nL 132nM dose at 72 hpf. Lyophilised IFN- γ-rel was reconstituted in PBS + 0.1% BSA (carrier protein) and PBS + 0.1% BSA was used as the vehicle control solution. Injections of recombinant zfVegfaa (Kingfisher Bioscience) were administered as single 1nL 0.25 ug/ul doses at 72 hpf (protein reconstituted as above).
Pacific blue tagged 500 kDa dextran (Fina Biosolutions) (dissolved at 5% w/v in DPBS) was injected at 1nL into injured larvae 2 hpi and the heart and pericardial fluid immediately imaged by heartbeat-synchronised light-sheet microscopy.
For propidium iodide intravital staining, 1nL 100μg/ml propidium iodide in DPBS was injected immediately following injury at 0.5 hpi. Larvae were then immediately imaged by heartbeat-synchronised light-sheet microscopy at 1 hpi. Injection of recombinant zfIFN-γ-rel (IFN-1.1) (Kingfisher Bioscience) was administered as a single 1nL 132nM dose at 72 hpf. Lyophilised IFN- γ-rel was reconstituted in PBS + 0.1% BSA (carrier protein) and PBS + 0.1% BSA was used as the vehicle control solution. Injections of recombinant zfVegfaa (Kingfisher Bioscience) were administered as single 1nL 0.25 ug/ul doses at 72 hpf (protein reconstituted as above).
Pacific blue tagged 500 kDa dextran (Fina Biosolutions) (dissolved at 5% w/v in DPBS) was injected at 1nL into injured larvae 2 hpi and the heart and pericardial fluid immediately imaged by heartbeat-synchronised light-sheet microscopy.