Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis
Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis
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DOI：
10.2144/02322st03
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发表时间：
2002-02-01
期刊：
影响因子：
2.7
通讯作者：
Cheng, KC
中科院分区：
文献类型：
--
作者：
Moore, JL;Aros, M;Cheng, KC
为了便于成年斑马鱼组织的分子分析，我们测试了8种不同的固定和脱钙条件，以获得适合PCR和组织免疫反应性的DNA，石蜡包埋和切片。尽管所有条件都导致良好的组织组织学和免疫细胞化学，但只有两种条件使DNA保持完整，如通过PCR所见。结果表明，斑马鱼固定在10%中性缓冲福尔马林或4%多聚甲醛，然后在0.5 M EDTA脱钙，是一种简单可靠的方法，允许分子实验和组织学进行相同的标本。通过Dietrich溶液的固定和脱钙允许PCR扩增250而不是1000 bp的DNA片段。因此，福尔马林或多聚甲醛固定，然后用EDTA脱钙的协议是广泛适用于各种脊椎动物组织时，优秀的组织学，免疫细胞化学，和基因型分析可能同时需要。
To facilitate the molecular analysis of tissues in adult zebrafish, we tested eight different fixation and decalcification conditions for the ability to yield DNA suitable for PCR and tissue immunoreactivity, following paraffin embedding and sectioning. Although all conditions resulted in good tissue histology and immunocytochemistry, only two conditions left the DNA intact as seen by PCR. The results indicate that zebrafish fixed in either 10% neutral buffered formalin or 4% paraformaldehyde, followed by decalcification in 0.5 M EDTA, is an easy and reliable method that allows molecular experiments and histology to be performed on the same specimen. The fixation and decalcification by Dietrich's solution permitted the PCR amplification of DNA fragments of 250 but not 1000 bp. Therefore, a protocol of formalin or paraformaldehyde fixation followed by decalcification with EDTA is broadly applicable to a variety of vertebrate tissues when excellent histological, immunocytochemical, and genotypic analyses may be simultaneously required.