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

IF 4.4 1区 生物学 Q1 BIOLOGY BMC Biology Pub Date : 2024-11-04 DOI:10.1186/s12915-024-02052-3
Carlos Guerrero-Hernández, Viraj Doddihal, Frederick G Mann, Alejandro Sánchez Alvarado
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Abstract

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.

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用于免疫染色和原位杂交的功能强大、用途广泛的新固定方案,可保护脆弱的组织。
背景:了解基因如何发挥愈合伤口和恢复失去的组织的功能对研究再生至关重要。整装原位杂交(WISH)是一种功能强大、应用广泛的技术,可用于观察不同生物系统中基因的表达模式。然而,用于 WISH 的现有透化样本方法会损伤或破坏脆弱的再生组织,从而阻碍此类实验的进行:结果:在此,我们介绍了一种在再生能力极强的刨叶虫 Schmidtea mediterranea 中进行原位杂交(ISH)和免疫染色的新方案。这种新的硝酸/甲酸(NAFA)方案与这两种检测方法兼容,并能防止表皮和再生胚泡退化。NAFA方案无需使用蛋白酶K消化就能实现这一目的,这可能会更好地保存抗原表位。我们的研究表明,NAFA 方案成功地在原位形成了色原和荧光信号,同时保留了动物的解剖结构。此外,在进行荧光原位杂交后,不同蛋白质的免疫染色与 NAFA 方案兼容。此外,该组织固定方案很容易适用于再生鳉鱼尾鳍,能产生更好的 ISH 信号,且背景极低:因此,NAFA 方案既能有效保护脆弱的受伤组织,又能促进探针和抗体穿透内部组织。此外,该固定方案还可用于再生鳍鱼的 WISH,这表明它将成为研究多种鱼类创伤反应和再生过程的重要技术。
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来源期刊
BMC Biology
BMC Biology 生物-生物学
CiteScore
7.80
自引率
1.90%
发文量
260
审稿时长
3 months
期刊介绍: BMC Biology is a broad scope journal covering all areas of biology. Our content includes research articles, new methods and tools. BMC Biology also publishes reviews, Q&A, and commentaries.
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