Effectiveness of fixation methods for wholemount immunohistochemistry across cellular compartments in chick embryos.
Effectiveness of fixation methods for wholemount immunohistochemistry across cellular compartments in chick embryos.
复制标题
鸡胚胎细胞区室整体免疫组织化学固定方法的有效性。
DOI：
10.1101/2024.03.23.586361
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Rogers,CrystalD
中科院分区：
文献类型：
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作者：
EcheverriaJr,CamiloV;Leathers,TessA;Rogers,CrystalD
固定方法的选择对免疫组化（IHC）后的组织形态和蛋白可视化有显著影响。在本研究中，我们比较了IHC前多聚甲醛（PFA）和三氯乙酸（TCA）固定对鸡胚的影响。我们的研究结果强调了验证固定方法对于准确解释免疫组化结果的重要性，这对抗体验证和组织特异性蛋白定位研究具有重要意义。我们发现，与PFA固定相比，TCA固定导致核更大、更圆。此外，TCA固定改变了亚细胞定位的外观和各种蛋白质的荧光强度，包括转录因子和细胞骨架蛋白。值得注意的是，TCA固定揭示了PFA固定可能无法进入的蛋白质定位结构域。这些结果强调了根据目标表位和模型系统优化固定方案的必要性，强调了生物学分析中方法学考虑的重要性。
The choice of fixation method significantly impacts tissue morphology and protein visualization after immunohistochemistry (IHC). In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation prior to IHC on chicken embryos. Our findings underscore the importance of validating fixation methods for accurate interpretation of IHC results, with implications for antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei compared to PFA fixation. Additionally, TCA fixation altered the appearance of subcellular localization and fluorescence intensity of various proteins, including transcription factors and cytoskeletal proteins. Notably, TCA fixation revealed protein localization domains that may be inaccessible with PFA fixation. These results highlight the need for optimization of fixation protocols depending on the target epitope and model system, emphasizing the importance of methodological considerations in biological analyses.