Click it edu cell proliferation kit for imaging alexa flour 647

To visualize newly deposited CaCO₃ of the skeleton, live animals were incubated with Calcein (Sigma) as described in [41 (link)]. To understand the spatial distribution and quantity of proliferating cells during juvenile sea urchin growth assays of cell proliferation were carried out using the Click-iT® EdU Alexa Flour® 555HCS (Life Technologies) and Click-iT™ EdU Cell Proliferation Kit for Imaging Alexa Flour™ 647 (Thermo Fisher Scientific) following [41 (link)]. Cells were quantified for analyzed images using ImageJ and R. Additional details of Calcein staining, proliferative staining and imaging can be found in Additional file 1: Methods.

In order to understand the spatial distribution of proliferating cells across the putative broad cell types, cell proliferation assays were carried out using Click-It EdU Cell Proliferation Kit for Imaging Alexa Flour 647 (Thermo Fisher Scientific). Larvae were treated with EdU at a final concentration of 10 μM in FSW and let to grow for 2 hr. Samples were fixed in 4 % PFA in FSW for 15 min (RT) and washed several times with PBST. PBST was removed, replaced by 100 % Methanol for 1 min (RT) and followed by several washes with PBST. After this step, one can continue with either developing the EdU signal or performing immunohistochemistry as described above. In order to develop the EdU signal, the Click-iT reaction mix was prepared according to the manufacturer’s guidelines. PBST was removed and the reaction mix was added to the samples for 30 min (RT). Larvae were washed several times with PBST, mounted and imaged using a Zeiss LSM 700 confocal microscope.