Methods for Staining Amyloid in Tissues: A Review

Abstract

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.

Hyperforin prevents β-amyloid neurotoxicity and spatial memory impairments by disaggregation of Alzheimer's amyloid-β-deposits

“…Congo red (CR) staining using 'alkaline CR methods' was performed as previously described. 24,25 Image analysis The ROS fluorescence was imaged with a LSM 5 Pascal Zeiss confocal microscope, and analyzed by Sigman Scan Pro software. 26 The fluorescence intensity quantification was carried out as the difference between the final fluorescence emitted by the neurons in the presence of the respective treatment and the background fluorescence of the cells without treatment of 10 pictures of three independent experiments.…”

Risk-adjusted manipulation of melphalan dose before stem cell transplantation in patients with amyloidosis is associated with a lower response rate

“…7 Patients whose disease was limited to cutaneous involvement, purpura, or carpal tunnel syndrome were excluded. The diagnosis of light-chain-related AL required evidence of a serum or urine monoclonal light chain or the presence of clonal plasma cells in the bone marrow.…”

Revealing molecular-level surface structure of amyloid fibrils in liquid by means of frequency modulation atomic force microscopy

“…While the structure of amyloid fibrils remains unclear, there are several defining features that are generally accepted. Amyloid fibrils commonly have a β-sheet-rich structure, which characteristically binds the histological dyes Thioflavin T and Congo red [11]. Their X-ray diffraction patterns show ~0.5 nm along, and ~1.0 nm reflections perpendicular to the fibril axis [12][13][14].…”