Special staining is used to visualize tissue structures, abnormal deposits, and pathogen components that are difficult to distinguish by HE staining. Its advantages include mature methodology, relatively low cost, and intuitive results, making it suitable for fibrosis evaluation, tumor differentiation, vascular lesion observation, metabolic disease assessment, and screening of infectious pathogens.

Keywords: special staining; Masson’s trichrome staining; reticular fiber staining; elastic fiber staining; PAS staining; lipid staining; pigment staining; pathogenic microorganism staining

1 Technical Positioning of Special Staining

1.1 Relationship with HE Staining

HE staining can display tissue architecture and basic cellular morphology, but it has limited ability to identify collagen fibers, reticular fibers, elastic fibers, glycogen, mucus, lipids, pigments, and some pathogens. Special staining uses specific dyes, redox reactions, metal impregnation, or histochemical reactions to make target components show characteristic colors or distribution patterns.

(1) Supplementary discrimination of tissue components

Collagen fibers, muscle fibers, reticular fibers, and elastic fibers often show similar color tones in HE sections. Special staining can improve the recognition of tissue structural layers.

(2) Auxiliary identification of abnormal deposits

Glycogen, mucus, hemosiderin, melanin, amyloid, and lipids may be difficult to accurately distinguish by routine staining. Special staining can be used for localization and preliminary characterization.

(3) Visualization of pathogens

Fungi, acid-fast bacilli, some bacteria, and spirochetes are not easily confirmed in HE sections. Special staining can improve pathogen detection and morphological recognition.

1.2 Relationship with Immunohistochemistry and Electron Microscopy

Special staining cannot replace immunohistochemistry, molecular testing, or electron microscopy, but it has high practical value in pathological screening and morphological evaluation. Immunohistochemistry is more suitable for identifying specific antigens, while electron microscopy is more suitable for observing ultrastructure. Special staining is better suited for displaying histochemical components, matrix structures, and the morphology of some pathogens.

(1) Pre-diagnostic screening

In fibrosis, mucus secretion, glycogen deposition, pigmentation, and infectious lesions, special staining can serve as a low-cost and rapid morphological supplementary method.

(2) Combination with immunohistochemistry

Tumor differentiation, stromal reaction evaluation, basement membrane disruption, and pathogen confirmation often require combined interpretation of special staining and immunohistochemistry.

(3) Combination with molecular testing

When special staining suggests infection, deposition, or metabolic abnormalities, culture, PCR, sequencing, mass spectrometry, or genetic testing may be further used to clarify etiology and classification.

Table 1 Main application directions of special staining

2 Special Staining for Fibers and Matrix

2.1 Collagen Fiber Staining

Collagen fibers are the main supporting components of connective tissue and have important diagnostic significance in fibrosis, scar formation, organ sclerosis, and tumor stromal reactions. In HE staining, collagen fibers are usually pink and are not clearly distinguished from muscle fibers and other stromal components. Therefore, Masson’s trichrome staining or other collagen staining methods are often used.

(1) Masson’s trichrome staining

Masson’s trichrome staining can distinguish collagen fibers, muscle fibers, and red blood cells. Common results include collagen fibers appearing blue or green, muscle fibers and cytoplasm appearing red, and red blood cells appearing orange-red. This method is suitable for evaluating liver cirrhosis, renal interstitial fibrosis, myocardial scarring, pulmonary fibrosis, and tumor stromal reactions.

(2) Assessment of fibrosis distribution

Collagen staining is used not only to evaluate total collagen content, but also to observe fiber distribution patterns. Bridging fibrosis, lobular architectural remodeling, encircling fibrous proliferation, and scar boundaries can be more clearly displayed by collagen staining.

(3) Differentiation of abnormal substances

Collagen fiber staining can help distinguish scar-like collagen from certain homogeneous deposits. If amyloid is suspected, Congo red staining and related methods should be used for further confirmation.

2.2 Reticular Fiber Staining

Reticular fibers are mainly composed of type III collagen-related structures and are commonly distributed in basement membranes, pericapillary areas, hematopoietic tissues, endocrine glands, and around glandular structures. Reticular fiber staining usually uses silver impregnation methods, which can clearly display tissue frameworks and fiber structures surrounding cell nests.

(1) Differentiation between carcinoma and sarcoma

Reticular fiber staining is important in distinguishing carcinoma from sarcoma. Carcinomas often form nested or cord-like structures, with fewer reticular fibers inside tumor nests; sarcoma cells may be surrounded by or intermingled with abundant reticular fibers.

(2) Observation of basement membrane disruption

In the evaluation of early invasion, reticular fiber staining can assist in observing basement membrane continuity. Disruption or interruption of the reticular basement membrane framework may support invasive growth.

(3) Evaluation of liver and lymphoid tissue architecture

Structural framework changes in chronic hepatitis, liver cirrhosis, and lymph node architectural destruction can be observed by reticular fiber staining. This staining helps evaluate focal necrosis, fibrosis severity, and tissue architectural remodeling.

2.3 Elastic Fiber Staining

Elastic fibers are mainly distributed in vascular walls, lung tissue, skin, and some connective tissues, and are responsible for stretching and recoil. Elastic fiber staining can show elastic fiber proliferation, rupture, fragmentation, loss, and abnormal arrangement.

(1) Analysis of vascular lesions

Arterial elastic laminae, vascular contours, and elastic layer destruction can be visualized by elastic fiber staining. Atherosclerosis, large-vessel vasculitis, vascular wall injury, and vascular lesions can be evaluated using this staining.

(2) Lung and skin lesions

In emphysema, elastic fibers in alveolar septa are destroyed. Elastic fiber changes are also prominent in cutis laxa, lichen sclerosus et atrophicus, and related lesions. Elastic fiber staining can assist in assessing the degree of structural damage.

(3) Tumor-related evaluation

Some elastofibroma-like lesions or vascular-origin lesions can be assisted by the distribution pattern of elastic fibers. Results should be interpreted together with HE morphology and immunohistochemistry.

Table 2 Application comparison of fiber-related special stains

3 Carbohydrate, Mucus, and Lipid Staining

3.1 PAS Staining

PAS staining oxidizes carbohydrate structures with periodic acid and reacts with Schiff reagent to display glycogen, neutral mucus, basement membranes, fungal cell walls, and some glycoprotein components. Positive structures usually appear magenta or reddish-purple.

(1) Glycogen deposition

PAS staining can be used to evaluate glycogen storage disease, hepatocellular glycogen changes, myocardial glycogen deposition, and intracellular glycogen in certain tumor cells. If glycogen identity needs confirmation, diastase digestion can be used.

(2) Basement membrane and glomerular lesions

PAS can display basement membranes, glomerular mesangial matrix, and glycoprotein components of vascular walls. It is commonly used in renal pathology and diabetic microvascular lesion evaluation.

(3) Fungal screening

Fungal cell walls are rich in polysaccharides, and PAS can show hyphae, spores, and some fungal structures. If higher sensitivity is required, GMS silver staining is often further used.

3.2 Mucus Staining

Mucus can be divided into epithelial secretory mucus and connective tissue mucinous substances, and can be further classified into neutral mucus and acidic mucus. Mucus staining is commonly used in adenocarcinoma, signet-ring cell carcinoma, mucinous degeneration, and gastrointestinal lesion analysis.

(1) AB-PAS staining

AB-PAS can simultaneously visualize acidic and neutral mucus. It is commonly used for gastrointestinal epithelial differentiation, mucinous cell carcinoma, and mucus secretion type evaluation. The distribution patterns of different mucus types can provide evidence for tumor classification and tissue origin assessment.

(2) Differentiation between signet-ring cells and foam cells

In gastric biopsies, signet-ring cells may sometimes resemble xanthoma cells or foam cells morphologically. Mucus staining can reveal intracellular mucus components and assist in identifying tumor-related mucus secretion.

(3) Differentiation between edema and mucinous degeneration

In skin, soft tissue, and stromal lesions, simple edema and mucinous degeneration may appear similar in HE sections. Acidic mucus staining can display stromal mucinous substances and improve the basis for differentiation.

3.3 Lipid Staining

Lipids are easily extracted by organic solvents during routine paraffin processing, so lipid staining usually requires frozen sections. Common methods include Sudan III, Sudan Black B, and Oil Red O.

(1) Fatty degeneration

Fatty degeneration in tissues such as liver, myocardium, and kidney can be shown by lipid staining to visualize lipid droplet distribution. This method is suitable for observing the extent of lipid deposition and intracellular lipid droplet status.

(2) Fat embolism

When fat embolism is suspected in the heart, brain, lung, kidney, or other tissues, lipid staining can help determine whether intravascular emboli contain lipid droplets.

(3) Differentiation of adipocytic tumors

Liposarcoma and other mesenchymal tumors may require combined evaluation using lipid staining, morphology, and immunohistochemistry. Lipid droplet positivity can provide evidence of adipocytic differentiation, but it cannot establish the diagnosis alone.

Table 3 Applicable scenarios for carbohydrate, mucus, and lipid staining

4 Pigment, Amyloid, and Pathogen Staining

4.1 Pigment Staining

Brown-yellow or brown-black pigments in tissues may be confused with each other in HE sections. Hemosiderin, melanin, formalin pigment, and exogenous pigments should be interpreted using special staining together with medical history.

(1) Hemosiderin staining

Prussian blue staining can display ferric iron-related pigments and is commonly used for evaluating old hemorrhage, hemochromatosis, hemosiderin deposition, and post-hemorrhagic tissue changes. A positive result indicates iron-related deposition, but interpretation should still consider tissue location and pathological background.

(2) Melanin staining

Fontana-Masson staining can display melanin and some reducing silver-positive substances. When used for melanoma, pigmented lesions, and tissue pigment characterization, results should be interpreted together with immunohistochemistry and clinical information.

(3) Logic of pigment differentiation

The focus of pigment staining is not simply observing color, but identifying pigment origin. Hemosiderin suggests hemorrhage- or iron metabolism-related changes, whereas melanin suggests melanocytic origin or pigment-producing lesions.

4.2 Amyloid Staining

Congo red staining is used to detect amyloid. Positive areas appear red or orange-red under ordinary light and can show characteristic birefringence under polarized light. This method is commonly used for amyloidosis screening and characterization of tissue deposits.

(1) Differentiation of deposits

Homogeneous eosinophilic deposits in HE sections may resemble collagen, fibrinoid necrosis, or other proteinaceous deposits. Congo red can be used to confirm whether amyloid is present.

(2) Verification of positive results

Amyloid type still requires further evaluation using immunohistochemistry, mass spectrometry, or relevant clinical data. Congo red positivity mainly indicates deposit identity and does not directly determine amyloid subtype.

4.3 Pathogenic Microorganism Staining

In infectious lesions, HE staining may show only inflammatory reactions and may not clearly identify the pathogen type. Special staining can improve the detection rate of fungi, acid-fast bacilli, and some bacteria.

(1) Fungal staining

PAS and GMS can both display fungal structures. PAS is suitable for preliminary visualization of organisms and tissue reactions, while GMS is more sensitive for fungal cell walls and is commonly used for necrotizing inflammation, granulomatous inflammation, and suspected fungal infection samples.

(2) Acid-fast staining

Acid-fast staining is used for detecting acid-fast pathogens such as Mycobacterium tuberculosis and some nontuberculous mycobacteria. A positive result supports acid-fast bacillary infection, but species confirmation requires culture or molecular testing.

(3) Gram staining

Gram staining can be used to observe bacterial morphology and Gram staining characteristics. It is suitable for purulent inflammation or tissues suspected of bacterial infection. When bacterial numbers are low in tissue sections, a negative result cannot completely exclude infection.

Table 4 Special stains for pigments, deposits, and pathogens

5 Staining of Muscle, Nerve, and Special Tissue Structures

5.1 Muscle Tissue Staining

Phosphotungstic acid hematoxylin (PTAH) can be used to display striated muscle structures, muscle fiber injury, and some fibrinoid substances. This method has certain application value in myocardial lesions, muscle injury, and striated structure observation.

(1) Myocardial injury

Myocardial fiber degeneration, necrosis, and striation changes can be observed with PTAH staining. Results should be interpreted together with HE staining, Masson staining, and clinical background.

(2) Muscle lesions

In skeletal muscle diseases, PTAH can assist in observing structural changes in muscle fibers. If specific myopathy classification is required, enzyme histochemistry, immunohistochemistry, and molecular testing should still be combined.

5.2 Nervous Tissue Staining

Neurofibrils and myelin structures are not easily recognized in HE sections. Special staining for nervous tissue can be used to observe nerve fiber injury, nervous system tumors, and demyelinating lesions.

(1) Neurofibrils

Neurofibril staining can display neuronal cell bodies, dendrites, axons, and related fiber structures, helping to observe nerve injury and neuronal structural changes.

(2) Myelin staining

Myelin-related staining can be used for demyelinating diseases, white matter lesions, and nervous tissue structural evaluation. Interpretation should consider lesion location, inflammatory cell distribution, and clinical information.

(3) Differentiation of nervous system tumors

Some nervous system tumors may resemble undifferentiated tumors in HE morphology. Nervous tissue special staining can provide auxiliary morphological evidence, but it cannot replace immunohistochemistry and molecular classification.

6 Staining Workflow and Quality Control

6.1 Pre-Staining Control

(1) Section quality

Paraffin sections should have uniform thickness and no obvious folds, cracks, or knife marks. Sections that are too thick can cause heavy staining background, while sections that are too thin may affect visualization of weakly positive components.

(2) Tissue position

The tissue should be located in an appropriate area of the slide to avoid incomplete reagent coverage during deparaffinization and staining. When multiple tissues are mounted on the same slide, each tissue area should be fully exposed to staining reagents.

(3) Complete deparaffinization

Incomplete deparaffinization of paraffin sections prevents staining reagents from evenly contacting the tissue, resulting in staining failure, localized weak staining, or patchy background abnormalities. The deparaffinization-to-water process should be sufficient and stable.

6.2 Staining Process Control

(1) Strictly follow timing and sequence

Special staining often depends on multiple steps such as oxidation, reduction, mordanting, differentiation, and counterstaining. Insufficient or excessive timing in any step may alter the final result.

(2) Adequate washing

Residual staining or reaction solution from the previous step may affect the next reaction. Insufficient washing often results in increased background, abnormal color, or unclear positive structures.

(3) Control staining reagent coverage

When drop-staining, the staining solution should completely cover the tissue without overflow, which can cause reagent waste and uneven staining. A hydrophobic barrier pen can be used around the tissue.

(4) Include controls

Positive controls and negative or digestion controls should be included whenever possible. Methods such as PAS-D, acid-fast staining, GMS, Prussian blue, and Congo red especially require controls to support result interpretation.

6.3 Result Interpretation Control

(1) Positive location

Interpretation should focus on whether the positive signal is located intracellularly, extracellularly, in the basement membrane, vascular wall, stroma, or pathogen structures. Color change alone does not necessarily have diagnostic significance.

(2) Background interference

Precipitates, residual staining reagents, endogenous pigments, and section contamination may cause false positivity. HE sections and control slides should be used to exclude technical interference.

(3) Integrated diagnosis

Special staining results should serve the pathological diagnostic chain. Final judgment requires integration of HE morphology, clinical information, immunohistochemistry, molecular testing, or pathogen detection results.

Table 5 Common problems in special staining and troubleshooting directions

7 Selection Pathway for Special Staining Methods

7.1 Selection by Target Structure

(1) Fibrosis and matrix structures

Masson’s trichrome staining is preferred for observing collagen fibers, reticular fiber staining for framework structures, and elastic fiber staining for vascular elastic laminae and elastic structures in lung and skin.

(2) Carbohydrates and mucus

PAS is selected to display glycogen, basement membrane, and fungal structures. AB-PAS is selected to distinguish acidic and neutral mucus. When glycogen is suspected, digestion controls can improve interpretation reliability.

(3) Lipids

Lipid detection should preferably use frozen sections and lipid staining methods such as Sudan III, Oil Red O, or Sudan Black B. Paraffin processing is usually unsuitable for preserving neutral fat.

(4) Pigments and pathogens

Prussian blue is selected for suspected iron deposition, Fontana-Masson for suspected melanin, Congo red for suspected amyloid, PAS or GMS for suspected fungi, and acid-fast staining for suspected acid-fast bacilli.

7.2 Selection by Lesion Question

(1) Organ sclerotic diseases

For liver cirrhosis, renal fibrosis, pulmonary fibrosis, and myocardial scarring, Masson staining can be prioritized. Reticular fiber staining can be added when tissue framework changes need evaluation.

(2) Tumor differentiation

For carcinoma versus sarcoma, mucinous tumors, melanocytic tumors, adipocytic tumors, and neurogenic tumors, reticular fiber, mucus, melanin, lipid, or nervous tissue-related staining can be selected according to the target component.

(3) Vascular lesions

For atherosclerosis, vasculitis, arteriovenous structural evaluation, and elastic layer destruction, elastic fiber staining can be selected. Masson staining and immunohistochemistry may be combined when vascular wall remodeling needs evaluation.

(4) Infectious lesions

When purulent inflammation, granulomatous inflammation, necrotizing inflammation, or giant cell reactions raise suspicion of infection, GMS, PAS, acid-fast staining, or Gram staining should be selected according to lesion morphology.

8 Related Reagent and Material Selection

Table 6 Product selection related to special staining

9 Frequently Asked Questions

9.1 Can special staining replace immunohistochemistry?

Not completely. Special staining mainly displays histochemical components and structural distribution, while immunohistochemistry detects specific antigen expression. The two often complement each other in tumor differentiation, infection evaluation, and tissue structure assessment.

9.2 What is mainly evaluated by Masson’s trichrome staining?

Masson’s trichrome staining is mainly used to show differences between collagen fibers and muscle fibers. It is commonly used for evaluating fibrosis, scars, sclerotic lesions, and tumor stromal reactions. Interpretation should focus on the distribution range, thickness, orientation, and tissue architectural remodeling of collagen.

9.3 Why does reticular fiber staining help distinguish carcinoma from sarcoma?

Reticular fiber staining shows the relationship between tumor cells and the stromal framework. Carcinomas often form nested or cord-like structures with few reticular fibers inside tumor nests, whereas sarcoma cells often have reticular fibers surrounding or interspersed among them.

9.4 Does PAS positivity always indicate glycogen?

No. PAS can show glycogen, neutral mucus, basement membranes, fungi, and various glycoprotein components. To confirm glycogen, diastase digestion or other histological evidence should be combined.

9.5 Why does lipid staining usually require frozen sections?

Lipids are easily extracted by organic solvents such as alcohol and xylene during paraffin processing, resulting in loss of lipid droplets. Frozen sections better preserve neutral fats and lipid droplet structures.

9.6 Both GMS and PAS can stain fungi. How should they be selected?

PAS can display fungi and tissue polysaccharide structures and is suitable for preliminary observation. GMS is more sensitive for fungal cell walls and is suitable for samples with few organisms, complex necrotic background, or suspected deep fungal infection.

9.7 Can positive pigment staining directly determine the disease type?

No. Pigment staining can help characterize pigment types, such as iron deposition or melanin, but disease type still requires integration of tissue location, HE morphology, clinical information, and necessary immunohistochemistry results.

The value of special staining lies in supplementing HE staining information through intuitive histochemical visualization. In practice, staining methods should be selected according to target structure, lesion question, and sample processing method. Standardized section preparation, complete deparaffinization, strict washing, appropriate controls, and integrated interpretation improve result reliability.

For more related articles, please see below:

[1] Masson’s Trichrome Stain (Masson’s Trichrome) Standard Operating Procedure (SOP)