Comparison of Histological Techniques to Visualize Iron in Paraffin-embedded Brain Tissue of Patients with Alzheimer’s Disease

Abstract

SummaryBetter knowledge of the distribution of iron in the brains of Alzheimer's disease (AD) patients may facilitate the development of an in vivo magnetic resonance (MR) marker for AD and may cast light on the role of this potentially toxic molecule in the pathogenesis of AD. Several histological iron staining techniques have been used in the past but they have not been systematically tested for sensitivity and specificity. This article compares three histochemical techniques and ferritin immunohistochemistry to visualize iron in paraffin-embedded human AD brain tissue. The specificity of the histochemical techniques was tested by staining sections after iron extraction. Iron was demonstrated in the white matter, in layers IV/V of the frontal neocortex, in iron containing plaques, and in microglia. In our hands, these structures were best visualized using the Meguro iron stain, a method that has not been described for iron staining in human brain or AD in particular. Ferritin immunohistochemistry stained microglia and iron containing plaques similar to the Meguro method but was less intense in myelin-associated iron. The Meguro method is most suitable for identifying iron-positive structures in paraffinembedded human AD brain tissue. (J Histochem Cytochem 61:785-792, 2013)

Methods for quantitative susceptibility and R2* mapping in whole post-mortem brains at 7T

“…Furthermore, microglial ferritin staining has been demonstrated in the frontal cortex of Alzheimer's disease [50] and in the deep layers of the motor cortex of ALS [24]. Since the majority of iron is bound to ferritin, studies have validated intracellular ferritin as a surrogate marker for iron content in tissue [50,51]. Shortcomings of immunostaining with respect to quantification are described below.…”

Evaluating Iron Flux in the Brain

“…These sections should then be left at RT for 30 min before the staining procedure is attempted. For paraffin embedded tissue, optimal staining outcomes have been noted for tissue fixed in neutral buffered formalin (1%, w/v) and sectioned at 5-10 μm [64]. As with most histological procedures for paraffin embedded tissue, sections require deparaffinizing and hydrating before staining.…”

Post-mortem T2*- weighted MRI imaging of cortical iron reflects severity of Alzheimer’s Disease

“…Tissue blocks were embedded in paraffin and serially cut into 8-μm and 20-μm-thick sections. Histochemical iron detection was done as previously described (Bulk et al, 2018;van Duijn et al, 2013). After deparaffinization, 20μm-thick sections were incubated for 80 minutes in 1% potassium ferrocyanide, washed, followed by 100 minutes incubation in methanol with 0.01 M NaN3 and 0.3% H2O2.…”

“…Histopathological stainings have been performed for: Aβ, tau tangles, iron and myelin. The same stainings have previously been investigated (Bulk et al, 2018;van Duijn et al, 2013), and the same semi-quantitative scoring system was used in this study. All substrates were assessed by two trained blinded observers (MB, BK).…”