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A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria

Carlos Guerrero-Hernández, View ORCID ProfileViraj Doddihal, View ORCID ProfileFrederick G. Mann Jr., View ORCID ProfileAlejandro Sánchez Alvarado
doi: https://doi.org/10.1101/2021.11.01.466817
Carlos Guerrero-Hernández
1Stowers Institute for Medical Research, Kansas City, MO, USA
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Viraj Doddihal
1Stowers Institute for Medical Research, Kansas City, MO, USA
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Frederick G. Mann Jr.
1Stowers Institute for Medical Research, Kansas City, MO, USA
2Howard Hughes Medical Institute, Kansas City, MO, USA
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Alejandro Sánchez Alvarado
1Stowers Institute for Medical Research, Kansas City, MO, USA
2Howard Hughes Medical Institute, Kansas City, MO, USA
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  • For correspondence: asa@stowers.org
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Abstract

Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression pattern of genes in different biological systems. Here we describe a new protocol for ISH and immunostaining in the planarian Schmidtea mediterranea. The new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both assays and prevents degradation of the epidermis or blastema. Instead of proteinase K digestion, formic acid treatment is used to permeabilize tissues and preserve 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. Further, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Finally, we demonstrate with high resolution confocal imaging that the regeneration blastema is preserved when using the new method. This new NAFA protocol will be a valuable technique to study the process of wounding response and regeneration.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 03, 2021.
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A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria
Carlos Guerrero-Hernández, Viraj Doddihal, Frederick G. Mann Jr., Alejandro Sánchez Alvarado
bioRxiv 2021.11.01.466817; doi: https://doi.org/10.1101/2021.11.01.466817
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A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria
Carlos Guerrero-Hernández, Viraj Doddihal, Frederick G. Mann Jr., Alejandro Sánchez Alvarado
bioRxiv 2021.11.01.466817; doi: https://doi.org/10.1101/2021.11.01.466817

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