Lumar v12 microscope

Melanoma cells were treated with PXDN, NTN4 or GLIS3-specific siRNAs or scrambled control siRNA as described above and labelled with CM-DiO or Dil as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates consisting of similar sized small aggregates from approximately 500–800 cells were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts were analyzed for the localization of melanoma cells using Lumar V12 Zeiss microscope as described previously [20 (link)]. Segments of embryos containing the melanoma cells were then washed in PBS, incubated overnight in 30% sucrose at 4°C, frozen and 20μm transverse sections cut using a cryostat. Sections were rinsed in PBS, mounted in fluorescent mounting media and imaged using a Zeiss Axio M1 microscope and AxioVision software.

Melanoma cells were treated with THBS1-specific siRNAs or scrambled control siRNA as described and labelled with CM-DiO or Dil as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates consisting of 50-400 cells were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts or cross sections were analyzed for the localization of melanoma cells using Lumar V12 Zeiss microscope.

We evaluated the impact of PEAR1 rs12041331 genotype on ex vivo endothelial cell migration using methods described previously [23 (link)]. Briefly, 10 primary HUVEC lines were randomly chosen within each genotype (4 rs12041331 major allele homozygotes [GG], 4 heterozygotes [GA], and 2 minor allele homozygotes [AA]) and split at passage 3 onto a gelatin-coated 6-well plate containing Endothelial Basal Media 2. Confluent HUVEC monolayers were scraped in a uniform manner using a P1000 pipette tip to generate the scratch, which was followed by replacement of growth media. Cells were photographed at 0 and 6 hours using an Axiocam MRc 5 camera (Carl Zeiss Microscopy, Pleasanton, California) mounted on a Lumar.V12 microscope (Carl Zeiss Microscopy, Pleasanton, California). The area of the scratch was measured using ImageJ [24 (link)] and used to calculate endothelial cell migration as described in the Statistical Analysis section.