Application Differences Among Highman, Bennhold, Puchtler, and Modified Methods in Congo Red Staining for Amyloid

Congo red staining is a classical method for identifying amyloid in histopathology and experimental histology. The differences among the Highman, Bennhold, Puchtler, and modified Congo red methods do not lie in their recognition of different types of amyloid protein, but in their staining environment, differentiation control, background handling, and stability of polarized light interpretation.

Keywords: Congo red staining; amyloid; Highman method; Bennhold method; Puchtler alkaline Congo red method; modified Highman method; modified Stores method; polarized light; apple-green birefringence; histopathological staining

1 Basic Logic of Congo Red Staining

1.1 Staining Basis of Amyloid

(1) Staining target

Congo red staining is mainly used to display amyloid in tissue sections. Amyloid refers to a class of abnormal protein deposits characterized by β-sheet structures. It may be distributed in vessel walls, interstitium, organ parenchyma, or around localized lesions.

(2) Microscopic appearance

After Congo red binds to amyloid, deposits usually appear red, orange-red, or brick-red under ordinary light microscopy. This appearance is suitable for morphological localization, but it cannot be used alone as confirmatory evidence.

(3) Polarized light interpretation

Typical apple-green birefringence under polarized light is an important criterion for identifying amyloid using the Congo red method. If only red staining is observed under ordinary light microscopy without typical birefringence, the effects of background staining, section thickness, differentiation conditions, or mounting status should be considered.

1.2 Technical Differences Among Congo Red Methods

(1) Staining system

Different Congo red methods differ in staining solution composition, pH environment, and differentiation system. The Bennhold method is closer to a classic basic display method, the Highman method emphasizes tissue localization and contrast, while the Puchtler alkaline method places greater emphasis on selective binding and background control.

(2) Background control

Collagen, elastic fibers, necrotic tissue, myxoid matrix, and pigment deposits may all interfere with observation under ordinary light microscopy. Samples with complex backgrounds are more suitable for the Puchtler alkaline Congo red method or modified Congo red methods.

(3) Method selection

Samples with obvious deposits can be displayed using the Bennhold or Highman method. Weakly positive, focal, or background-complex samples are more suitable for the Puchtler alkaline method, modified Highman method, or modified Stores method.

Table 1 Key Points for Interpreting Congo Red Staining Results

Interpretation Level	Observation Content	Positive Features	Main Risks
Ordinary light microscopy	Color and distribution of deposits	Red, orange-red, or brick-red amorphous deposits	Interference from collagen, necrotic tissue, or background red staining
Polarized light	Birefringence pattern	Apple-green birefringence	Section thickness, overstaining, or mounting may affect interpretation
Tissue localization	Deposits in vessel walls, interstitium, or parenchyma	Corresponds to suspicious areas on HE staining	Misinterpretation caused by tissue folds or nonspecific deposits
Method consistency	Results from different methods or counterstaining	Positive areas appear consistently	Insufficient differentiation may lead to false-positive tendency

2 Application Differences Among the Highman, Bennhold, and Puchtler Methods

2.1 Bennhold Congo Red Method

(1) Method positioning

The Bennhold method is a classical Congo red staining system that emphasizes the direct staining ability of Congo red for amyloid deposits. It is suitable for basic display and teaching observation of samples with obvious deposits.

(2) Staining characteristics

Under ordinary light microscopy, the Bennhold method mainly focuses on red or orange-red deposits. Its operational logic is relatively clear, but background control depends more on differentiation conditions and operator experience.

(3) Applicable scenarios

When amyloid deposition is abundant, the tissue background is relatively simple, and the main purpose is routine screening or basic observation, the Bennhold method has good applicability. If the sample contains abundant collagen, marked necrosis, or strong background red staining, polarized light observation and other modified methods should be used for confirmation.

2.2 Highman Congo Red Method

(1) Method positioning

The Highman method places greater emphasis on tissue contrast and morphological localization based on classical Congo red staining, making it suitable for routine observation of amyloid deposits in tissue sections.

(2) Staining characteristics

The advantage of the Highman method is that positive deposits can be relatively clearly distinguished from surrounding tissue structures under ordinary light microscopy. However, the result still requires confirmation by polarized light birefringence and should not be judged positive based only on red deposits.

(3) Applicable scenarios

The Highman method is suitable for routine verification of suspected amyloid deposits in pathological tissues. For samples with small deposits or complex tissue backgrounds, the modified Highman method or Puchtler alkaline method is usually more helpful in improving interpretation reliability.

2.3 Puchtler Alkaline Congo Red Method

(1) Method positioning

The Puchtler alkaline Congo red method is a widely used modified Congo red system. Its core feature is the use of an alkaline environment and a salt-alcohol system to enhance the selective binding of Congo red to amyloid and reduce nonspecific background staining.

(2) Staining characteristics

The Puchtler method usually provides good background control. Positive deposits appear red to orange-red under ordinary light microscopy and can show relatively typical apple-green birefringence under polarized light.

(3) Applicable scenarios

The Puchtler alkaline Congo red method is more suitable for clinical pathological confirmation, weakly positive deposit screening, and analysis of tissues with complex backgrounds. It is particularly valuable for samples from the kidney, liver, spleen, myocardium, gastrointestinal tract, and vessel walls.

Table 2 Application Differences Among the Highman, Bennhold, and Puchtler Methods

Method	Technical Positioning	Main Advantages	Main Limitations	Applicable Scenarios
Bennhold Congo red method	Classical basic staining system	Clear operational logic; suitable for observing obvious deposits	Background control depends on differentiation; weakly positive samples are harder to interpret	Obvious amyloid deposits, basic screening, teaching observation
Highman Congo red method	Emphasizes tissue contrast and morphological localization	Relatively clear localization under ordinary light microscopy; good tissue layering	Still requires polarized light confirmation; differentiation must be controlled in complex backgrounds	Routine tissue section verification and pathological morphological localization
Puchtler alkaline Congo red method	Alkaline Congo red system with stronger selectivity	Good background control; relatively stable polarized light performance	Requires good control of pH, differentiation, section thickness, and workflow consistency	Weakly positive deposits, complex tissues, clinical pathological confirmation

3 Technical Characteristics of Modified Congo Red Methods

3.1 Modified Highman Method

(1) Purpose of modification

The modified Highman method is usually optimized around staining stability, background reduction, and readability under ordinary light microscopy. Its purpose is not to change the principle by which Congo red recognizes amyloid, but to improve the distinction between positive deposits and background tissue.

(2) Application characteristics

The modified Highman method is suitable for samples with complex tissue structures, obvious background red staining, or insufficient contrast using the conventional Highman method. It can be used for rechecking unclear results obtained with the routine Highman method.

(3) Interpretation focus

When using the modified Highman method, insufficient differentiation should be avoided because it may enhance background red staining, while over-differentiation should also be avoided because it may weaken weakly positive deposits. Positive areas should be mutually verified with suspicious HE regions and polarized light birefringence.

3.2 Modified Stores Congo Red Method

(1) Method positioning

The modified Stores Congo red method is an optimized system for improving amyloid display and background control, and it can complement other Congo red methods.

(2) Application characteristics

The modified Stores method is suitable for samples with abundant background tissue, scattered deposits, or a need for improved staining consistency. Its advantage lies in improving the distinction between tissue background and positive deposits.

(3) Quality control

The modified Stores method should emphasize positive controls, section thickness, staining time, and differentiation conditions. If birefringence in the positive control weakens, the staining solution status and dehydration/mounting process should be checked first.

3.3 Other Supplementary Systems

(1) Methanol Congo red system

The methanol Congo red system can be used for auxiliary observation or method comparison under specific tissue background conditions. Its results should be interpreted together with conventional Congo red staining and polarized light results, and it should not be used alone as confirmatory evidence.

(2) Methyl violet method

The methyl violet method can be used as a supplementary method for amyloid staining and compared with Congo red results. Its value mainly lies in providing deposit observations under a different staining system, rather than replacing polarized light confirmation.

(3) Basic Congo red staining

Ordinary Congo red staining solution is suitable for method pilot experiments, basic observation, or self-established differentiation systems. If it is used for amyloid confirmation, positive controls and polarized light observation must be strictly included.

Table 3 Application Focus of Modified Congo Red Methods and Supplementary Systems

Method	Technical Focus	Suitable Samples	Interpretation Focus
Modified Highman method	Improves tissue contrast and staining stability	Samples with complex backgrounds or insufficient contrast using routine Highman method	Consistency between red deposits and polarized light birefringence
Modified Stores Congo red method	Improves background control and recognition of positive deposits	Scattered deposits and structurally complex tissues	Positive controls, section thickness, and differentiation conditions
Methanol Congo red system	Adjusts dye penetration and tissue dehydration environment	Specific experimental conditions or supplementary validation	Should not replace polarized light confirmation
Methyl violet method	Supplementary staining method for amyloid	Method comparison or auxiliary observation	Cross-verification with Congo red results

4 Method Selection and Result Control

4.1 Logic of Method Selection

(1) Samples with obvious deposits

For samples with obvious amorphous eosinophilic deposits on HE staining and relatively simple tissue backgrounds, the Bennhold, Highman, or Puchtler method can all be used for initial display. Formal interpretation should still include polarized light observation.

(2) Weakly positive or focal deposit samples

For samples with limited deposits, focal distribution, or mild vessel wall involvement, the Puchtler alkaline Congo red method or modified Congo red methods should be preferred. Such samples are more sensitive to section thickness, staining time, and differentiation conditions.

(3) Samples with complex backgrounds

Samples rich in collagen, obvious myxoid matrix, marked necroinflammation, or abundant pigment deposits should not be judged based only on red staining under ordinary light microscopy. Background differential staining, positive controls, and polarized light birefringence should be analyzed together.

4.2 Control of Experimental Conditions

(1) Section thickness

Overly thick sections may increase background staining and nonspecific birefringence, while overly thin sections may make weakly positive deposits difficult to display. Section thickness should be kept consistent in Congo red staining for amyloid to improve batch-to-batch comparability.

(2) Degree of differentiation

Insufficient differentiation can increase background red staining, while excessive differentiation may weaken weakly positive deposits. The Puchtler and modified methods are relatively sensitive to differentiation steps, so appropriate differentiation intensity should be determined using positive controls.

(3) Mounting and polarized light observation

Dehydration, clearing, and mounting can affect both ordinary light microscopy color and polarized light performance. If ordinary light microscopy is positive but polarized light is not typical, slide preparation and observation conditions should be checked first.

Table 4 Selection of Congo Red Methods for Different Experimental Needs

Experimental Need	Preferred Method	Auxiliary Method	Reason for Selection
Initial screening of obvious amyloid deposits	Bennhold method or Highman method	Puchtler method	Direct and intuitive operation; suitable for rapid display of deposit areas
Clinical pathological confirmation	Puchtler alkaline Congo red method	Modified Highman method	Better background control and more reliable polarized light interpretation
Weakly positive or focal deposits	Puchtler method or modified Stores method	Modified Highman method	Improves distinction between deposits and background tissue
Collagen-rich tissues	Puchtler method	Modified Congo red method; collagen background staining	Reduces interference from collagen or interstitial background
Methodological comparison	Bennhold, Highman, and Puchtler methods in parallel	Modified methods for rechecking	Facilitates comparison of staining intensity, background, and polarized light effect
Teaching and basic observation	Bennhold method or ordinary Congo red staining	Polarized light observation	Helps understand the basic morphology of Congo red-positive deposits

5 Congo Red Staining Products and Auxiliary Differential Reagents

Table 5 Product Selection for Congo Red Amyloid Staining and Auxiliary Differential Staining

Product Category	Cat. No.	Product Name	Method or System	Specification / Grade	Applicable Scenario
Core staining system	A1508461	Amyloid Staining Solution (Bennhold Congo Red Method)	Bennhold Congo red method	BioReagent,for microscopy,Biological Stain	Basic display, routine screening, and teaching observation of obvious amyloid deposits
Core staining system	A1508468	Amyloid Staining Solution (Highman Congo Red Method)	Highman Congo red method	BioReagent,for microscopy,Biological Stain	Amyloid localization in tissue sections, morphological observation, and routine pathological verification
Core staining system	A1508466	Amyloid Staining Solution (Puchtler Alkaline Congo Red Method)	Puchtler alkaline Congo red method	BioReagent,for microscopy,Biological Stain	Clinical pathological confirmation, weakly positive deposits, complex tissue backgrounds, and polarized light interpretation
Modified staining system	A1508467	Amyloid Staining Solution (Modified Highman Congo Red Method)	Modified Highman Congo red method	BioReagent,for microscopy,Biological Stain	Samples in which routine Highman method has insufficient contrast, obvious background red staining, or requires improved recognition of positive deposits
Modified staining system	S774555	Amyloid Staining Solution (Modified Stores Congo Red Method)	Modified Stores Congo red method	BioReagent, Biological Stain, for microscopy	Scattered deposits, complex background samples, and rechecking or comparison of different Congo red methods
Supplementary staining system	A1508465	Amyloid Staining Solution (Crystal Violet Method)	Methyl violet method	BioReagent,for microscopy,Biological Stain	Supplementary method for amyloid staining, used for comparison with Congo red results or auxiliary observation
Basic Congo red system	C1516057	Congo Red Staining Solution (1%)	Conventional Congo red staining	BioReagent,Biological Stain,for microscopy,1%	Basic Congo red staining, method pilot experiments, non-diagnostic observation, and self-established differentiation systems
Special Congo red system	M1520403	Methanol Congo Red Staining Solution (0.5%)	Methanol Congo red system	BioReagent,Biological Stain,for microscopy,0.5%	Auxiliary observation, method comparison, or optimization of Congo red systems under specific experimental conditions
Nuclear counterstain	N743376	Nuclear Fast Red Staining Solution	Nuclear counterstaining	BioReagent, Biological Stain, for microscopy, 0.1%	Displays tissue structure and nuclear background after Congo red staining
Nuclear counterstain	N1516052	Nuclear Fast Red Staining Solution (0.2%)	Nuclear counterstaining	BioReagent,Biological Stain,for microscopy,0.2%	Congo red-stained sections requiring enhanced nuclear background contrast
Hematoxylin counterstain	M774769	Mayer hematoxylin staining solution	Hematoxylin counterstaining	BioReagent, Biological Stain, for microscopy	Tissue structure counterstaining; helps observe the relationship between deposit areas and tissue structures
Hematoxylin counterstain	I1507737	Improved Harris Hematoxylin Staining Solution	Hematoxylin counterstaining	BioReagent,Suitable for microbiology,for microscopy	Nuclear counterstaining or tissue structure background display in Congo red staining workflows
Bluing treatment	B1194496	Dako Bluing Buffer	Hematoxylin bluing	BioReagent, Suitable for Immunohistochemistry(IHC)	Bluing after hematoxylin counterstaining to improve nuclear display stability
Tissue morphology control	E489517	Ematoxylin-Eosin Stain	HE staining		HE morphological prescreening of amyloid deposit areas and adjacent-section comparison
Mucus background differentiation	A1507852	Alcian Blue Staining Solution (pH2.5)	Acidic mucus staining	BioReagent,for microscopy,Biological Stain	Helps exclude interference from acidic mucopolysaccharides or myxoid matrix in deposit interpretation
Mucus background differentiation	A774857	Alcian Blue Staining Solution (pH 1.0)	Strong acidic sulfated mucus staining	BioReagent, Biological Stain, for microscopy	Differentiation of strongly acidic mucus background, suitable for glandular or mucus-rich tissues
Mucus background differentiation	I1508464	Improved Hale Colloid Iron Polysaccharide Staining Solution	Mucopolysaccharide staining	BioReagent,for microscopy,Biological Stain	Used for mucopolysaccharide background differentiation to avoid confusion between myxoid matrix and amyloid deposits
Mucus background differentiation	M1508531	Mucicarmine Staining Solution	Mucus staining	BioReagent,for microscopy,Biological Stain	Used for differentiating mucinous substances and helping exclude mucus deposit interference
Glycogen/PAS background differentiation	G774821	Glycogen D-PAS Staining Solution (Amylase Digestion)	PAS differentiation	BioReagent, for microscopy, Biological Stain	Helps distinguish glycogen, PAS-positive background, and non-amyloid deposits
Basement membrane background differentiation	B774812	Methen Amine Silver Staining Solution (PASM)	Basement membrane staining	BioReagent, Biological Stain, for microscopy	Basement membrane background analysis in kidney tissue, assisting localization of renal amyloid deposits
Collagen background differentiation	V774853	Van Gieson Staining Solution	Collagen fiber background staining	BioReagent, Biological Stain, for microscopy	Helps distinguish collagen red staining from Congo red-positive deposits
Collagen background differentiation	I774852	Modified Van Gieson Staining Solution	Collagen fiber background staining	BioReagent, Biological Stain, for microscopy	Used for interstitial and collagen background differentiation, suitable for reviewing complex background samples
Collagen background differentiation	I774801	Modified Masson Trichrome Staining Solution	Collagen/muscle fiber differentiation	BioReagent, Biological Stain, for microscopy	Collagen-rich tissue background analysis, assisting interpretation of Congo red results in complex tissues
Connective tissue background differentiation	G774552	Goldner Tricolor Staining Solution	Connective tissue staining	BioReagent, Biological Stain, for microscopy	Used for connective tissue background evaluation and assisting assessment of interstitial deposit areas
Reticular fiber background differentiation	M774808	Reticular Fibre Staining Solution (Gomori)	Reticular fiber staining	BioReagent, Biological Stain, for microscopy	Assists deposit localization in liver, spleen, bone marrow, and other tissues
Reticular fiber background differentiation	M774851	Reticular Fibre Staining Solution (Gordon-Sweets)	Reticular fiber staining	BioReagent, for microscopy, Biological Stain	Displays reticular fiber framework and helps determine the relationship between deposits and interstitial structures
Elastic fiber background differentiation	V1518609	Verhöeff Elastic Fiber Staining Solution (Eosin Counterstain)	Elastic fiber staining	BioReagent,Biological Stain,for microscopy	Displays elastic fiber background in vessel walls and assists interpretation of vascular amyloid deposits
Elastic fiber background differentiation	V1518552	Verhöeff Elastic Fiber Staining Solution (Orange G Counterstain)	Elastic fiber staining	BioReagent,Biological Stain,for microscopy	Used for vascular structure and elastic fiber background differentiation, suitable for vessel wall deposit samples
Elastic fiber background differentiation	L1518590	Lichen Red Elastic Fiber Staining Solution	Elastic fiber staining	BioReagent,Biological Stain,for microscopy	Helps exclude interference from vascular wall elastic fibers in Congo red interpretation
Pigment background differentiation	M774546	Masson-Fontana Melanin Staining Solution	Melanin staining	BioReagent, Biological Stain, for microscopy	Analysis of pigmented tissue background to avoid pigment deposits affecting deposit observation
Pigment background differentiation	A1508695	Melanin-Lipofuscin Staining Solution (Schmorl's Method)	Pigment background staining	BioReagent,for microscopy,Biological Stain	Used for melanin or lipofuscin background differentiation and helps exclude pigment interference
Calcium salt background differentiation	C774862	Calcium salt staining solution (Von, Kossa, silver nitrate)	Calcium salt staining	BioReagent, Biological Stain, for microscopy	Calcium deposit differentiation, avoiding confusion between calcified lesions and deposition diseases
Calcium salt background differentiation	C774863	Calcium Staining Solution (Modified Alizarin Red S Method)	Calcium salt staining	BioReagent,for microscopy,Biological Stain	Used for calcium salt background differentiation and assisting analysis of complex deposition lesions
Iron deposit background differentiation	P1508698	Prussian Blue Staining Solution (DAB Enhancement Method)	Iron staining	BioReagent,for microscopy,Biological Stain	Differentiation of hemosiderin or iron deposits, helping exclude pigment-background interference

6 Common Problems and Interpretation Pitfalls

6.1 False-Positive Risks

(1) Collagen and interstitial background

Tissues rich in collagen fibers may show background red staining or nonspecific color interference, especially when differentiation is insufficient. In this situation, deposit morphology, tissue localization, and polarized light performance should be emphasized.

(2) Necrotic and inflammatory areas

Necrotic tissue, inflammatory exudates, or tissue debris may adsorb dye and form focal red staining. If the positive area lacks a typical amorphous deposit morphology and does not show typical apple-green birefringence under polarized light, interpretation should be cautious.

(3) Section folds and uneven thickness

Section wrinkles, folds, or locally excessive thickness can create optical artifacts and affect polarized light observation. When suspicious birefringence appears, slide preparation factors should be excluded first, and adjacent sections should be restained for comparison.

6.2 False-Negative Risks

(1) Limited amount of deposits

Early or focal amyloid deposits may stain weakly and be difficult to identify under ordinary light microscopy. In such cases, suspicious HE regions should be examined at high magnification and rechecked with polarized light and modified methods.

(2) Over-differentiation

Excessive differentiation may weaken Congo red-positive deposits, especially in weakly positive samples. If the positive control also becomes lighter, abnormal differentiation time or differentiation solution conditions should be considered.

(3) Poor staining solution status

Aged staining solution, inappropriate pH, or unstable salt-alcohol systems may reduce staining selectivity and birefringence performance. For Puchtler and modified methods, staining solution status should be regularly verified using positive controls.

Table 6 Common Problems and Optimization Directions in Congo Red Staining

Problem	Possible Cause	Effect on Results	Optimization Direction
Strong background red staining	Insufficient differentiation, collagen-rich tissue, overstaining	Increased risk of false positivity	Optimize differentiation time; use Puchtler or modified methods
Pale positive deposits	Over-differentiation, reduced staining solution activity, limited deposits	Increased risk of false negativity	Use positive controls; shorten differentiation or replace staining solution
Atypical polarized light	Excessively thick sections, poor mounting, overstaining	Affects confirmatory interpretation	Control section thickness; optimize mounting and observation conditions
Large batch-to-batch variation	Inconsistent staining solution status, time, temperature, or operation	Reduced reproducibility	Standardize workflow and include positive tissue controls
Positive ordinary light microscopy but negative polarized light	Nonspecific red staining or abnormal technical conditions	High risk of misinterpretation	Restain and interpret together with modified methods and positive controls

The core differences among the Highman, Bennhold, Puchtler, and modified Congo red methods lie in staining selectivity, background control, and stability of polarized light interpretation. The Bennhold method is suitable for basic display, the Highman method emphasizes tissue localization, the Puchtler alkaline method is more suitable for complex tissues and confirmatory testing, and modified methods improve interpretation reliability for weakly positive or background-complex samples.

For more related articles, please see below:

[1] Fluorescent staining of frozen sections for amyloid (AMYLOID) thioflavine T

[2] Histological and Cytological Section Staining: Principles and Methodological Essentials for Routine Pathology Stains and Structural Special Stains