Meguro Reiko, Asano Yoshiya, Odagiri Saori, Li Chengtai, Iwatsuki Hiroyasu, Shoumura Kazuhiko
Department of Anatomy, Hirosaki University School of Medicine, Hirosaki, Japan.
Arch Histol Cytol. 2007 Apr;70(1):1-19. doi: 10.1679/aohc.70.1.
We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the Perls and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistry. The Perls method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The Perls or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H(2)O(2) and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.
我们回顾了非血红素铁组织化学方法，特别关注其潜在的化学原理。术语“非血红素铁”包括铁络合物的异质种类，其中铁与低分子量有机碱和蛋白质的结合比血红素（铁原卟啉复合物）中的铁更松散。非血红素铁在稀酸溶液中释放出来，可通过珀尔斯法、特恩布尔法和其他使用铁螯合剂的方法用于传统组织化学，这些方法依赖于不溶性铁化合物的产生。血红素铁的释放需要用强氧化剂处理，因此传统组织化学无法对其进行染色。实验室研究中最常用的珀尔斯法主要对三价铁染色，但也能对一些二价铁染色，而特恩布尔法对后者具有特异性。尽管在切片上进行的特恩布尔法无法对二价铁染色或仅对铁大量积累的组织部分染色，但体内灌注特恩布尔法显示二价铁广泛分布，尤其是在溶酶体中。用于电子显微镜的DAB/银/金方法可增强珀尔斯或特恩布尔反应。硫化铁法以及用H₂O₂和DAB对氧化还原活性铁进行染色也适用于电子显微镜。尽管上述组织化学方法在通过传统光学和电子显微镜观察铁方面具有优势，但铁的定量估计并不容易。最近依赖于Fe²⁺对荧光二价金属指示剂的淬灭以及二价金属螯合剂对其的去淬灭的方法，使得能够对分离的活细胞中可螯合的Fe²⁺进行定量估计。
