We used the transgenic green fluorescent zebrafish Tg fli:enhanced GFP strain for our xenograft studies. The experiments were carried out according to the standard guidelines approved by the local Institutional Committee for Animal Welfare of Leiden University. Zebrafish extravasation assay was performed as previously described (104 ). In brief, PaTu-S mCherry cells were pretreated with TGF-β for 2 days, and approximately, 400 cells were injected at the ducts of Cuvier into the zebrafish embryos at 48 h postfertilization. Then the injected zebrafish embyos were kept at 33 °C for 4 days after injection. The latter temperature is a compromise for both the fish and cells. Thereafter, the fish were fixed with 4% paraformaldehyde and imaged by an inverted SP5 confocal microscopy (Leica Microsystems). The number of invasive cell clusters (more than five cells were defined as a cluster) between the vessels in the caudal hematopoietic tissue region was counted.
Inverted sp5 confocal microscopy
Manufactured by Leica
The Inverted SP5 confocal microscopy system is a high-performance imaging tool designed for advanced microscopy applications. It features a flexible, modular design that allows for the integration of various detection and illumination modules to suit diverse research needs. The system's core function is to provide high-resolution, high-sensitivity imaging capabilities for a wide range of biological and materials science applications.
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3 protocols using inverted sp5 confocal microscopy
1
Tracking Tumor Cell Extravasation in Zebrafish
2
Zebrafish Extravasation Assay for Cancer Cell Invasion
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Zebrafish extravasation assays were performed as previously described [44 (link)]. The experiments were carried out according to the standard guidelines that were approved by the local Institutional Committee for Animal Welfare of Leiden University. The fish were fixed with 4% paraformaldehyde (PFA) four days after injection with mCherry-labeled MDA-MB-231 cells into the Duct of Cuvier and imaged by inverted SP5 confocal microscopy (Leica Microsystems). The numbers of cancer cells that had invaded into the avascular tail fin area, which is rich in collagen, were counted (Supplementary Fig. S5E). The experiments were repeated twice in biologically independent experiments, and at least 25 injected embryos were included for quantification.
3
Xenograft Assay in Transgenic Zebrafish
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We used the transgenic green fluorescent zebrafish Tg fli:enhanced green fluorescent protein (EGFP) strain for our xenograft studies. The experiments were carried out according to the standard guidelines approved by the local Institutional Committee for Animal Welfare of the Leiden University. Zebrafish extravasation assay was performed as was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted May 17, 2021. ; https://doi.org/10.1101/2021.05.14.444203 doi: bioRxiv preprint 24 previous described (100) . In brief, PaTu-S mCherry cells were pretreated with TGF-β for 2 days and approximately 400 cells were injected at the ducts of Cuvier into the zebrafish embryos at 48 h-post-fertilization (48-hpf) . Then the injected zebrafish embryos were kept at 33°C for 4 days after injection. The latter temperature is a compromise for both the fish and cells. Thereafter, the fish were fixed with 4% paraformaldehyde and imaged by an inverted SP5 confocal microscopy (Leica Microsystems). The number of invasive cell clusters (more than 5 cells were defined as a cluster) between the vessels in the caudal hematopoietic tissue region were counted.
The copyright holder for this preprint (which this version posted May 17, 2021. ; https://doi.org/10.1101/2021.05.14.444203 doi: bioRxiv preprint 24 previous described (100) . In brief, PaTu-S mCherry cells were pretreated with TGF-β for 2 days and approximately 400 cells were injected at the ducts of Cuvier into the zebrafish embryos at 48 h-post-fertilization (48-hpf) . Then the injected zebrafish embryos were kept at 33°C for 4 days after injection. The latter temperature is a compromise for both the fish and cells. Thereafter, the fish were fixed with 4% paraformaldehyde and imaged by an inverted SP5 confocal microscopy (Leica Microsystems). The number of invasive cell clusters (more than 5 cells were defined as a cluster) between the vessels in the caudal hematopoietic tissue region were counted.
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