Whole mount images of limbs for Alcian blue/Alizarin red staining, limb size measurements, in situ hybridization, bead experiments, cell death assays, body axis analysis, and eye position and pigmentation analysis were taken on an SZX10 light microscope (Olympus, Tokyo, Japan) using a DP73 CCD camera (Olympus). The microscope was equipped with CellSense software (CellSense version 1.12, Olympus Corporation).
EdU-stained stage 45 larval limbs were imaged using a Zeiss Lightsheet Z.1 (College of Arts and Science Imaging Center, University of Kentucky). Refer to the work of Purushothaman et al. (2019) (link) for detailed protocol of light-sheet microscopy for axolotl limb buds. Zen software (Zeiss) was used for imaging, and samples were excited using 561- and 488-nm lasers. Arivis vision4D software (Arivis) was used for image processing. For total limb volume calculations, an object mask was hand drawn at each z-plane on the basis of the DAPI signal to outline the limb. Red cell aggregate volume and total limb volume were calculated using the previously standardized protocols, and volume values in cubic micrometers and voxel counts were given as outputs.
EdU-stained stage 45 larval limbs were imaged using a Zeiss Lightsheet Z.1 (College of Arts and Science Imaging Center, University of Kentucky). Refer to the work of Purushothaman et al. (2019) (link) for detailed protocol of light-sheet microscopy for axolotl limb buds. Zen software (Zeiss) was used for imaging, and samples were excited using 561- and 488-nm lasers. Arivis vision4D software (Arivis) was used for image processing. For total limb volume calculations, an object mask was hand drawn at each z-plane on the basis of the DAPI signal to outline the limb. Red cell aggregate volume and total limb volume were calculated using the previously standardized protocols, and volume values in cubic micrometers and voxel counts were given as outputs.