Versatile Dual Mounting Enables Larval Zebrafish Imaging Across Microscope Configurations

Larval zebrafish are often mounted laterally to ensure consistent anatomical positioning and to standardize imaging of body axes across early development. However, this conventional approach often tethers sample orientation to a single microscope configuration and limits optical accessibility. We present a mounting protocol for larval zebrafish that enables optical access from both dorsal and ventral orientations while preserving lateral sample position. This approach uses common laboratory consumables to establish a mounting platform that eliminates any need to remount samples between the use of upright and inverted microscopes. By establishing a hydrophobic seal, mounted embryos can be inverted with ease to access the sample from either orientation. A seamless transition here facilitates reliable identification and longitudinal tracking of the same biological region of interest across microscope configurations. This protocol is broadly applicable to live imaging experiments requiring flexibility in imaging geometry, minimal sample handling, and high reproducibility. Key features • Dual-configuration mounting setup can be established from standard, low-cost consumables in any laboratory setting. • Preserves lateral mounting in agarose for consistent anatomical positioning and experimental continuity. • Enables seamless transition between upright and inverted microscopes without the need for specimen remounting.