Sharay E Setti1, James Raymick1, Joseph Hanig2, Sumit Sarkar3. 1. Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA. 2. Office of Testing & Research, Center for Drug Evaluation Research/FDA, Silver Spring, MD, USA. 3. Division of Neurotoxicology, National Center for Toxicological Research, U.S. Food & Drug Administration, Jefferson, AR, USA. Electronic address: Sumit.Sarkar@FDA.hhs.gov.
Abstract
BACKGROUND: Congo Red (CR) has been used for its binding affinity to amyloid fibrils for the better part of a century. Recently, our laboratory has demonstrated its ability to bind to tau protein as well. NEW METHOD: Here we describe a novel methodology for fast, thorough, whole-brain labeling of amyloid plaques with CR via perfusion. We tested five different variants which altered the volume of CR, the speed of perfusion, and the solution CR was solubilized in to determine the best results. RESULTS AND CONCLUSION: We determined that intra-cardiac perfusion of animals with 0.5 % CR in 100 ml of 50 % ethanol or perfusion with 0.5 of CR in 100 ml of 10 % neutral buffer formalin both perfused at a rate of 30 ml/min for 3.3 min resulted in the clearest CR labeling, with little to no background noise. Both variants were compatible with subsequent immunolabeling procedures for NU-1, as well as Ferritin and GFAP. Compared to traditional CR plaque labeling methodology, this new method allows for quick whole brain CR-labeling. This reduces the amount of time from days to mere minutes. It also reduces potential for variability that would result from staining slides in batches. Thus, CR-perfusion is a rapid, thorough method that can be utilized to rapidly stain amyloid in the rodent brain. Published by Elsevier B.V.
BACKGROUND:Congo Red (CR) has been used for its binding affinity to amyloid fibrils for the better part of a century. Recently, our laboratory has demonstrated its ability to bind to tau protein as well. NEW METHOD: Here we describe a novel methodology for fast, thorough, whole-brain labeling of amyloid plaques with CR via perfusion. We tested five different variants which altered the volume of CR, the speed of perfusion, and the solution CR was solubilized in to determine the best results. RESULTS AND CONCLUSION: We determined that intra-cardiac perfusion of animals with 0.5 % CR in 100 ml of 50 % ethanol or perfusion with 0.5 of CR in 100 ml of 10 % neutral buffer formalin both perfused at a rate of 30 ml/min for 3.3 min resulted in the clearest CR labeling, with little to no background noise. Both variants were compatible with subsequent immunolabeling procedures for NU-1, as well as Ferritin and GFAP. Compared to traditional CR plaque labeling methodology, this new method allows for quick whole brain CR-labeling. This reduces the amount of time from days to mere minutes. It also reduces potential for variability that would result from staining slides in batches. Thus, CR-perfusion is a rapid, thorough method that can be utilized to rapidly stain amyloid in the rodent brain. Published by Elsevier B.V.