National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, Launceston, Tasmania, Australia.
J Fish Dis. 2013 Oct;36(10):831-9. doi: 10.1111/jfd.12078. Epub 2013 Feb 6.
Formaldehyde-based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross-linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral-buffered formalin and seawater Davidson's, formaldehyde-based fixatives commonly used in fish histopathology, were compared to formalin-free commercial fixatives PAXgene®, HistoChoice™MB* and RNAlater™. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.
甲醛基固定剂通常用于组织病理学，尽管它们的使用存在一些明显的缺点。甲醛通过共价交联蛋白质来固定组织，这一过程已知会掩盖抗原表位，从而降低广泛用于疾病诊断的免疫组织化学染色的强度。此外，甲醛固定极大地限制了从固定标本中回收 DNA 和 mRNA 的能力，不利于下游的分子分析。尽管需要互补方法，如原位杂交（ISH）和聚合酶链反应（PCR）来确认引起阿米巴性鳃病（AGD）的病原体 Neoparamoeba perurans 的存在，但已经可以通过对鳃组织的组织学检查可靠地诊断 AGD。随着分子技术越来越普遍地用于病原体鉴定，需要调整标本采集和保存方法，以便组织学和分子生物学都可以用于诊断相同的样本。本研究采用通用方法评估了 5 种不同的大西洋鲑（Salmo salar L.）鳃组织固定剂。与鱼类组织病理学中常用的中性缓冲福尔马林和海水 Davidson's 福尔马林基固定剂相比，评估了福尔马林自由的商业固定剂 PAXgene®、HistoChoice™MB*和 RNAlater™。通过一系列用于证明 AGD 的分析来评估每种固定剂，包括常规组织化学染色、免疫组织化学染色、ISH 和 DNA 提取，随后进行 PCR。这 5 种固定剂都适合于大西洋鲑鳃的组织学检查，海水 Davidson's 提供了最佳的组织病理学结果。在所评估的固定剂中，海水 Davidson's 和 PAXgene®被证明与分子生物学技术最兼容。它们都能从固定和包埋的标本中获得良好的 DNA 回收率、数量和完整性。这两种固定剂都能很好地保存组织和细胞形态，同时允许对相同的标本进行分子分析，这使得海水 Davidson's 和 PAXgene®似乎是大西洋鲑鳃 AGD 诊断和研究的最佳固定方法。
