A powerful and versatile new fixation protocol for immunostaining and in situ hybridization that preserves delicate tissues.

Background: Understanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments.

Results: Here, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background.

Conclusions: Thus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species.