Modification of methods to use Congo-red stain to simultaneously visualize amyloid plaques and tangles in human and rodent brain tissue sections

Metab Brain Dis. 2020 Dec;35(8):1371-1383. doi: 10.1007/s11011-020-00608-0. Epub 2020 Aug 27.


Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aβ) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aβ as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aβ. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.

Keywords: Alzheimer disease; Amyloid plaques; Congo red; Human brain; Neurofibrillary tangle; Transgenic rat.

MeSH terms

  • Alzheimer Disease / metabolism
  • Alzheimer Disease / pathology
  • Animals
  • Brain / metabolism*
  • Brain / pathology
  • Brain Chemistry / physiology
  • Coloring Agents / analysis
  • Coloring Agents / metabolism*
  • Congo Red / analysis
  • Congo Red / metabolism*
  • Humans
  • Mice
  • Mice, Transgenic
  • Neurofibrillary Tangles / chemistry
  • Neurofibrillary Tangles / metabolism*
  • Neurofibrillary Tangles / pathology
  • Optical Imaging / methods
  • Plaque, Amyloid / chemistry
  • Plaque, Amyloid / metabolism*
  • Plaque, Amyloid / pathology
  • Rats
  • Rodentia
  • Staining and Labeling / methods*


  • Coloring Agents
  • Congo Red