Tokuda Kazuhiro, Baron Byron, Kuramitsu Yasuhiro, Kitagawa Takao, Tokuda Nobuko, Morishige Naoyuki, Kobayashi Masaaki, Kimura Kazuhiro, Nakamura Kazuyuki, Sonoda Koh-Hei
Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, 755-8505, Japan.
Department of Systems Biochemistry in Pathology and Regeneration, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, 755-8505, Japan.
Jpn J Ophthalmol. 2018 Jul;62(4):481-490. doi: 10.1007/s10384-018-0592-7. Epub 2018 Apr 24.
Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson's fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions.
Experimental design.
Four fixative formulations (4% paraformaldehyde, Davidson's fixative, modified Davidson's fixative and an in-house fixative - TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared.
TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson's fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily.
It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.
有多种固定剂可供使用，但许多并不适合视网膜的组织形态学保存。研究人员特别关注视网膜组织学研究，这类研究通常使用戴维森固定剂而非4%甲醛（FA）。然而，后者有其局限性。本研究的目的是制备一种能使视网膜更接近体内状态的新型固定剂。
实验设计。
在视网膜上测试了四种固定剂配方（4%多聚甲醛、戴维森固定剂、改良戴维森固定剂和一种内部固定剂——TB - Fix），并比较了它们对组织形态学以及选定抗原标志物免疫组化染色的影响。
与FA、戴维森固定剂及其改良版本相比，苏木精 - 伊红染色后，TB - Fix显著改善了形态细节，最重要的是消除了神经纤维层的海绵状外观以及胞体（包括穆勒细胞）的肿胀。用TB - Fix或FA固定的视网膜样本在视网膜神经元和神经胶质细胞的免疫组织学染色中显示出可比的结果。重要的是，用FA固定的全眼球形状塌陷并导致人工视网膜脱离，而用TB - Fix固定的眼球避免了变形和脱离。此外，我们发现当用于固定已知容易从培养板上脱离的HEK293细胞时，TB - Fix也能防止细胞从培养板上脱离。
结果表明，TB - Fix在视网膜形态和化学成分的保存方面有全面的改善。
