Congo red is not a drug with a therapeutic target in this study. It is a histochemical dye that specifically binds to crossed β-pleated sheet structures found in misfolded protein aggregates (amyloid-like structures) [1]
.

To investigate if aggregates in mutant cells have misfolded β-sheet secondary structures, a straightforward screening method is to use Congo red histochemical staining. The histochemical dye Congo red exhibits a unique binding affinity for beta-sheet structures that overlap. By attaching to proteins in non-native conformations, wild-type HSPB1 should preserve protein homeostasis and stop substrate aggregation. Nevertheless, the T139M mutant fails to perform this function and instead causes misfolded proteins to accumulate, which Congo red targets for insertion between β-sheet structures. A straightforward method to investigate if aggregates in mutant cells have misfolded β-sheet secondary structures is to utilize Congo red histochemical staining [1].

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Detection of Congophilic Aggregates in Mutant HSPB1-Expressing Cells: HeLa cells were transfected with plasmids expressing either the mutant HSPB1-T139M or wild-type HSPB1. Twenty-four hours after transfection, cells were fixed with 10% formalin and stained with 1% Congo red for 5 minutes, followed by destaining with 0.01% potassium hydroxide in 50% ethanol. Coverslips were dehydrated through graded ethanol concentrations, mounted, and examined by fluorescence microscopy under a rhodamine filter. Cells expressing the mutant HSPB1-T139M displayed varying sizes of intracytoplasmic aggregates with congophilic properties (showing red fluorescence under rhodamine optics), while wild-type HSPB1-overexpressing cells did not display congophilia. This indicates that Congo red can specifically identify misfolded protein aggregates with β-pleated sheet secondary structures formed by the mutant HSPB1 protein [1]
.

Congo Red Staining Protocol for Detecting Protein Aggregates: HeLa cells were grown on coverslips and transfected with mutant or wild-type HSPB1 constructs. After 24 hours, cells were fixed with 10% formalin for 10 minutes. The fixed cells were then stained with 1% Congo red solution for 5 minutes. Destaining was performed using 0.01% potassium hydroxide in 50% ethanol. The coverslips were dehydrated through a graded ethanol series (increasing concentrations), mounted in mounting medium, and examined under fluorescence microscopy using a rhodamine filter. Cells containing misfolded protein aggregates with β-pleated sheet structures showed positive Congo red staining (congophilic aggregates) visualized as red fluorescence [1]
.

Metabolism / Metabolites
Congo red (an azo dye derived from benzidine) and 2-azofluorene (a derivative of 2-aminofluorene) were reduced after overnight incubation with rat intestinal bacterial suspensions. High-performance liquid chromatography and ultraviolet spectroscopy confirmed the presence of benzidine in the Congo red incubation extract. …In the presence of a mitochondrial post-activation system, the Congo red incubation extract was mutagenic to Salmonella typhimurium TA1538, but untreated Congo red was not mutagenic. …Reducive pretreatment using cecal bacteria can extend the application of the Ames assay in screening for potential mutagens.

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[1]. A novel p.T139M mutation in HSPB1 highlighting the phenotypic spectrum in a family. Brain Behav. 2017 Jul 21;7(8):e00774.

Congo red is an indicator dye that is blue-violet at pH 3.0 and red at pH 5.0. It contains 3,3'-(biphenyl-4,4'-diyldiazepine-2,1-diyl)bis(4-aminonaphthalene-1-sulfonate). Its function is similar to that of 3,3'-(biphenyl-4,4'-diyldiazepine-2,1-diyl)bis(4-aminonaphthalene-1-sulfonic acid). Congo red is the sodium salt of benzidine diazobis-1-naphthylamine-4-sulfonic acid; a diazo dye that is red in alkaline solutions and blue in acidic solutions, primarily used as an indicator and biological staining agent. It is also an acidic dye used to detect hydrochloric acid in gastric contents. Furthermore, it is used in histological examinations for amyloidosis.

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Role in This Study: Congo red was used as a histological tool to investigate the pathogenicity of a novel mutation (p.T139M) in the HSPB1 gene. The study hypothesized that this mutation might impair protein degradation function, resulting in the formation of misfolded protein aggregates. Congo red staining was employed to determine if the aggregates formed in cells expressing the mutant HSPB1 protein displayed amyloidogenic properties (i.e., contained β-pleated sheet structures) [1]
.
- Mechanism of Staining: Congo red is a diazo dye that has the ability to bind specifically to crossed β-pleated sheet structures found in amyloid fibrils. When bound to such structures, it exhibits characteristic green birefringence under polarized light or red fluorescence under rhodamine optics, allowing for the visualization of protein aggregates [1]
.
- Significance of Findings: The presence of Congo red-positive aggregates only in cells expressing the mutant HSPB1-T139M, but not in cells expressing wild-type HSPB1, provided evidence that this novel mutation causes protein misfolding and aggregation. This supported the pathogenicity of the mutation and suggested that the disease mechanism involves the accumulation of misfolded proteins [1]
.
- Proposed as a Screening Tool: The authors proposed that Congo red histochemical stain may serve as a simple screening tool for investigating whether aggregates in cells expressing mutant proteins have misfolded β-pleated sheet secondary structures [1]
.
- Staining Characteristics: Under the experimental conditions, Congo red-positive aggregates appeared as varying sizes of intracytoplasmic red-fluorescent structures when examined under rhodamine optics. These aggregates were observed as single or multiple inclusions within the cytoplasm of HeLa cells expressing the mutant HSPB1 protein [1]
.
- Source: Congo red used in this study was purchased from Sigma-Aldrich [1]
.

C32H22N6NA2O6S2

696.6632

696.083

573-58-0

11313

Pink to red solid powder

0.995 g/mL at 25 °C

>360 °C(lit.)

10.787

2

12

5

48

1180

0

IQFVPQOLBLOTPF-UHFFFAOYSA-L

InChI=1S/C32H24N6O6S2.2Na/c33-31-25-7-3-1-5-23(25)29(45(39,40)41)17-27(31)37-35-21-13-9-19(10-14-21)20-11-15-22(16-12-20)36-38-28-18-30(46(42,43)44)24-6-2-4-8-26(24)32(28)34;;/h1-18H,33-34H2,(H,39,40,41)(H,42,43,44);;/q;2*+1/p-2

disodium;4-amino-3-[[4-[4-[(1-amino-4-sulfonatonaphthalen-2-yl)diazenyl]phenyl]phenyl]diazenyl]naphthalene-1-sulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20 mg/mL (~28.71 mM)
H2O : ~1 mg/mL (~1.44 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)