Lsm 710 fcs confocal microscope

Transgenic zebrafish strains used in this study were Tg(fli1a:EGFP)^y1 [28 (link)], Tg(fli1a:H2B-mCherry)^uq37bh [43 (link)], and Tg(fli1aep:ERK-KTR-Clover)^uq39bh [32 (link)]. Larvae/embryos were anaesthetised in 0.08 mg/mL tricaine and mounted as previously described [44 (link)]. Zebrafish live-imaging was conducted using either an Olympus MVX fluorescent microscope, Zeiss LSM 710 FCS confocal microscope, Zeiss Elyra 780 confocal microscope, Nikon Yokogawa CSU-W1 spinning disc confocal microscope, or the Zeiss Z. 1 lightsheet fluorescent microscope.

Neonatal mice (P6) were killed by decapitation and their diaphragm was collected and fixed in 4% formaldehyde. The tissue was blocked and permeabilized overnight in PBS containing 5% donkey serum and 0.1% Triton-X (Sigma). Antibodies used were rabbit anti-LYVE-1 (11–034, AngioBio), goat anti-Fn (N-20, Santa Cruz Biotechnology inc.) and AlexaFluor 488 nm and 594 nm-conjugated secondary antibodies (Invitrogen). Tissues were mounted in Vectashield (Vector) for confocal imaging. Whole-mount z-stack images were acquired with a LSM 710 FCS confocal microscope with a 10x 0.3NA EC Plan-Neofluar objective lense with the ZEN 2010 software (all Zeiss) and processed with Photoshop CS5 (Adobe) and ImageJ software (NIH). Mice used in this study were bred and housed in the animal facility of ETH Zurich. Experiments were performed in accordance with the animal protocol 131/2014 approved by the local veterinary authorities (Kantonales Veterinäramt Zürich).

Back skin samples were embedded in OCT (Leica Biosystems, Newcastle, UK) and frozen in liquid nitrogen. 10-μm frozen sections were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT), washed in PBS and incubated with blocking solution (5% donkey serum, 0.2% BSA and 0.3% Triton X-100 in PBS) for 1 h at RT. Next, the sections were stained with primary antibodies overnight at 4°C and, after several washes, incubated with secondary antibodies for 30 min at RT. Primary antibodies were as follows: rat anti-CD31 (BD Biosciences, San Jose, CA, USA), rabbit anti-LYVE-1 (Angiobio, Del Mar, CA, USA), goat anti-LYVE-1 (R&D Systems), rat anti-CD68 (Abcam, Cambridge, MA, USA), goat anti-podoplanin (R&D Systems), rabbit anti-cytokeratin 15 (Abcam), rat anti-Foxp3 (eBioscience, San Diego, CA, USA), and goat anti-Prox1 (R&D systems). Secondary antibodies (all from Thermo Fisher, San Jose, CA, USA) were as follows: donkey anti-rabbit Alexa Fluor 488, 594 or 647, donkey anti-rat Alexa Fluor 488 or 594, donkey anti-goat Alexa Fluor 488 or 594, and chicken anti-goat Alexa Fluor 647. Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) was used for nuclear staining. Immunofluorescence images were acquired by an Axioskop 2 mot plus microscope (Carl Zeiss, Jena, Germany) and Z stacks of images were obtained using a Zeiss LSM 710 FCS confocal microscope.

For MCs ablation, embryos of Tg(–5.2lyve1b:DsRed2);Tg(pdgfrb:GFP) were laterally mounted in 1% low-melting point agarose at 57 hpf. An aISV with migrating LEC was chosen randomly, a GFP-positive MC was identified using 488 nm laser. MCs located ahead of migrating route were ablated using a two-photon laser at 790 nm (Mai Tai, Spectr-Physics Millenia PRO). Control ablations were performed as above but the adjacent area to the pdgfrb⁺ cell targeted with the two-photon laser. For aISV ablation, Tg(–5.2lyve1b:DsRed2);Tg(flt:YFP);Tg(pdgfrb:GFP) embryos were prepared as described above and an aISV, with LEC migrating along, was ablated using the two-photon laser targeting the connection point of dorsal longitudinal anastomotic vessel and aISV as well as proximal end of the aISV at the connection point to the DA. Larvae were imaged before and after ablation with a Zeiss LSM 710 FCS confocal microscope, which was followed by either time-lapse imaging for around 5 hours or follow-up confocal imaging at 3 dpf.

Mice were sacrificed, hair was removed with depilation cream and ears were harvested and split into two halves along the cartilage. Ear tissues or lymph nodes were fixed for two hours in 4% PFA at 4° C, washed for 1 hour in PBS and incubated in blocking solution (5% normal donkey serum, 1% BSA, 0.01% Triton-X 100 in PBS) for 4 hours at room temperature. Subsequently, samples were incubated overnight at room temperature with primary antibodies in blocking solution: rabbit anti-RFP (1:300, Rockland), goat anti-LYVE-1 (1:200, R&D Systems), goat anti-Prox1 (1:200, R&D Systems). After extensive washes in PBS, samples were incubated for 2 hours at room temperature with AlexaFluor 488, 594 or 647- conjugated secondary antibodies raised in donkey (1:200, Invitrogen). After at least 2 hours of washes in PBS, samples were mounted with Vectashield mounting media (Vector) on glass slides. Whole mount z-stacks were acquired using an LSM 710 FCS confocal microscope equipped with a 10x 0.3 NA EC Plan-Neofluar objective using ZEN software (Zeiss), and were processed with ImageJ software.