Histological Staining Methods for Identifying Hepatitis B Virus: Comparison of HE, Orcein, Aldehyde Fuchsin, Victoria Blue, Immunohistochemistry, and In Situ Hybridization

Identification of hepatitis B virus (HBV) in liver tissue should not rely on a single staining result. HE staining can indicate the pathological background of chronic hepatitis, interface hepatitis, hepatocyte degeneration, fibrosis, and “ground-glass hepatocytes,” but it cannot directly prove HBV infection. Orcein, aldehyde fuchsin, and Victoria blue staining can display HBsAg-related inclusions or specific cytoplasmic changes. Immunohistochemistry can localize HBsAg and HBcAg and is the main method for confirming HBV-related antigen expression in tissue sections. In situ hybridization can further display HBV nucleic acid signals and is used for studies of viral replication and tissue localization.

Keywords: hepatitis B virus; HBV histological staining; HBsAg; HBcAg; immunohistochemistry; orcein staining; aldehyde fuchsin staining; Victoria blue staining; in situ hybridization

1 Basic Positioning of HBV Histological Staining

1.1 Questions that histological staining can answer

(1) Whether hepatitis B-related liver tissue injury is present

HE staining can show portal inflammation, interface hepatitis, hepatocyte ballooning degeneration, necrosis, portal tract inflammation, and fibrosis. These changes support a background of chronic viral hepatitis, but they are not specific for HBV.

(2) Whether HBsAg-related cytoplasmic changes are present

When HBsAg accumulates extensively in hepatocytes, ground-glass hepatocytes may form. Orcein, aldehyde fuchsin, and Victoria blue staining can display such intracytoplasmic HBsAg-related material to some extent, but the results are affected by background components, cholestasis, copper-binding proteins, and staining conditions.

(3) Where viral antigens are located

Immunohistochemistry can directly display the distribution of HBsAg and HBcAg in hepatocytes. HBsAg is often seen in cytoplasmic or membrane-like distribution, while HBcAg may show nuclear positivity, cytoplasmic positivity, or mixed nuclear-cytoplasmic positivity. Different patterns can help assess infection status and viral replication activity.

(4) Whether viral nucleic acids are present in tissue

In situ hybridization can detect HBV DNA or RNA localization in tissue and is suitable for studying the viral genome, replication intermediates, or transcriptional activity. Although it is not a traditional histochemical stain, it provides spatial localization at the tissue section level.

Table 1 Basic comparison of HBV histological detection methods

Method	Detection target	Main positive finding	Specificity	Application positioning
HE staining	Liver tissue morphology	Inflammation, necrosis, fibrosis, ground-glass hepatocytes	Low	Evaluates hepatitis background and tissue injury pattern
Orcein method	HBsAg-related cytoplasmic material and some copper-binding proteins	Brownish-red to purplish-red granular or block-like cytoplasmic positivity in hepatocytes	Medium to low	Traditional screening for HBsAg-related inclusions
Aldehyde fuchsin method	HBsAg-related material, elastic fibers, and some mucin-like components	Purple to deep-purple cytoplasmic positivity	Medium to low	Auxiliary display of ground-glass hepatocytes and specific cytoplasmic inclusions
Victoria blue method	HBsAg-related cytoplasmic material and elastic structures	Blue to blue-purple cytoplasmic positivity	Medium	Auxiliary display of HBsAg-related changes and tissue structures
HBsAg immunohistochemistry	HBsAg protein	Cytoplasmic, membranous, or granular positivity	High	Confirms expression of HBV surface antigen in liver tissue
HBcAg immunohistochemistry	HBcAg protein	Nuclear, cytoplasmic, or nuclear-cytoplasmic positivity	High	Evaluates viral replication-related antigen expression
HBV in situ hybridization	HBV DNA/RNA	Nuclear, cytoplasmic, or mixed nucleic acid signal	High	Tissue localization of viral nucleic acids and replication research

2 HE Staining

2.1 Method function

(1) Assessment of pathological background

HE staining is the basic method for evaluating liver biopsy, resected liver tissue, or autopsy liver tissue. It can show portal inflammation, interface hepatitis, lobular inflammation, hepatocyte necrosis, ballooning degeneration, apoptotic bodies, cholestasis, and fibrotic structural changes.

(2) Observation of ground-glass hepatocytes

In some cases of chronic HBV infection, hepatocyte cytoplasm may appear pale, homogeneous, finely granular, or ground-glass-like. This change is often associated with HBsAg accumulation in the endoplasmic reticulum, but HE can only suggest the morphological feature and cannot serve as direct evidence of HBV infection.

(3) Basis for grading and staging liver disease

HE staining can be used to assess inflammatory activity and necrosis. However, fibrosis staging usually requires combination with fibrosis-related stains such as Masson, reticulin staining, or Sirius Red.

2.2 Interpretation points

(1) Inflammatory activity

Portal inflammation, interface hepatitis, lobular necrosis, and spotty necrosis should be observed. Chronic hepatitis B may show varying degrees of inflammatory activity, but these changes can also occur in other viral hepatitis, autoimmune hepatitis, and drug-induced liver injury.

(2) Hepatocyte morphology

Ground-glass hepatocytes may suggest HBsAg accumulation, but similar cytoplasmic changes may be affected by fixation, metabolic changes, or other cytoplasmic inclusions. Therefore, when suspected ground-glass changes are observed on HE, HBsAg immunohistochemistry or auxiliary histochemical staining should be used for confirmation.

(3) Fibrotic background

Chronic HBV infection can cause portal tract expansion, bridging fibrosis, and cirrhotic nodule formation. HE can preliminarily identify structural abnormalities, but precise fibrosis evaluation should be combined with dedicated fiber staining.

3 Orcein Method

3.1 Method principle

(1) Staining basis

The orcein method is a traditional histochemical staining method that can display inclusion-like material associated with HBsAg in hepatocyte cytoplasm. Positive areas are often seen in the cytoplasm of ground-glass hepatocytes and may appear brownish-red, reddish-brown, or purplish-red in granular, block-like, or diffuse patterns.

(2) Histological significance

In liver tissue from chronic HBV infection, orcein-positive cells often suggest accumulation of HBsAg-related material in hepatocytes. Its value lies in helping identify ground-glass hepatocytes that are not sufficiently obvious on HE and in serving as a traditional screening method to assist interpretation of HBV-related changes.

(3) Sources of nonspecificity

Orcein is not an HBsAg-specific antibody stain and may also display copper-binding proteins or other cytoplasmic components. Therefore, orcein positivity alone cannot confirm HBV infection and should be interpreted together with serology, HBsAg immunohistochemistry, or molecular detection.

3.2 Application features

(1) Advantages

The orcein method is relatively simple and can be applied to routine paraffin sections. It is suitable for assisting the display of HBsAg-related cytoplasmic changes in traditional pathology workflows. For typical ground-glass hepatocytes, this method can enhance recognition of positive cytoplasmic structures.

(2) Limitations

Its sensitivity and specificity are both lower than immunohistochemistry. In liver tissue, cholestasis, copper deposition-related components, or other cytoplasmic protein deposits can increase interpretive interference. A positive result only suggests the possibility of HBV-related changes and should not be used as the final confirmatory basis.

(3) Applicable scenarios

It is suitable for traditional histochemical screening under resource-limited conditions, teaching demonstrations, preliminary observation of HBsAg-related cytoplasmic changes, and morphological comparison with immunohistochemistry results.

4 Aldehyde Fuchsin Method

4.1 Method principle

(1) Staining basis

The aldehyde fuchsin method can display elastic fibers, some mucin-like substances, and certain inclusion-like components in hepatocytes. In HBV-related liver tissue, this method can make HBsAg-related cytoplasmic inclusions appear purple to deep purple.

(2) Relationship with HBsAg-related changes

When HBsAg accumulates in hepatocytes and produces ground-glass changes, aldehyde fuchsin staining can show corresponding cytoplasmic positive areas. Its positive distribution usually has a certain correspondence with ground-glass hepatocytes or HBsAg immunohistochemistry-positive areas.

(3) Staining specificity issue

Aldehyde fuchsin is not an HBV antigen-specific method. Elastic fibers, some mucin-like components, or other tissue components may also be stained. Therefore, interpretation should be based on hepatocyte localization, tissue morphology, and immunohistochemistry results.

4.2 Application features

(1) Advantages

Aldehyde fuchsin can serve as a traditional histochemical method to display HBsAg-related cytoplasmic inclusions. Its positive color is relatively clear, and it can also reflect some background tissue structures.

(2) Limitations

Because this method can stain multiple tissue components, background control and localization are important. If the positive signal is not located in hepatocyte cytoplasm or is inconsistent with the morphology of ground-glass cells, it should not be directly interpreted as HBsAg accumulation.

(3) Applicable scenarios

It is suitable for traditional pathological observation of HBV-related liver tissue, comparison with the orcein method, or as an auxiliary stain when immunohistochemistry is unavailable. Final confirmation should still rely on HBsAg/HBcAg immunohistochemistry.

5 Victoria Blue Method

5.1 Method principle

(1) Staining basis

The Victoria blue method can display elastic fibers and some cytoplasmic material related to HBsAg. In liver tissue, HBsAg-related cytoplasmic inclusions may appear blue to blue-purple and are often seen in ground-glass hepatocytes.

(2) Tissue localization features

The key point is to determine whether the positive signal is located in hepatocyte cytoplasm, whether it appears granular, clumped, or diffuse, and whether it corresponds to ground-glass changes on HE. If blue staining is mainly located in vascular walls, interstitium, or elastic structures, it should be interpreted as elastic fiber staining background rather than HBV-related positivity.

(3) Relationship with elastic fiber staining

Victoria blue itself is also a commonly used elastic fiber stain. Therefore, in liver tissue, it may simultaneously display vascular wall elastic fibers and HBsAg-related cytoplasmic components. Interpretation must distinguish hepatocyte cytoplasmic positivity from stromal elastic structure positivity.

5.2 Application features

(1) Advantages

Victoria blue can display some HBsAg-related cytoplasmic changes as blue or blue-purple signals and is helpful for identifying certain ground-glass hepatocytes. Compared with HE, it makes specific intracytoplasmic staining signals more prominent.

(2) Limitations

This method is still an auxiliary histochemical stain and cannot replace HBsAg immunohistochemistry. Because it can also stain elastic fibers, vascular structures, stromal background, and cytoplasmic positivity in liver tissue must be strictly distinguished.

(3) Applicable scenarios

It is suitable for comparison of traditional histochemical methods, auxiliary identification of ground-glass hepatocytes, and cases where simultaneous observation of liver tissue structural background is needed. Confirmation of HBV infection still requires specific antigen or nucleic acid detection.

Table 2 Comparison of three traditional histochemical methods

Method	Main display target	Typical positive finding	Advantages	Limitations
Orcein method	HBsAg-related cytoplasmic material and some copper-binding proteins	Brownish-red to purplish-red hepatocyte cytoplasm	Relatively simple operation; suitable for traditional screening	Insufficient specificity; may be interfered with by copper-binding proteins
Aldehyde fuchsin method	HBsAg-related inclusions, elastic fibers, and some mucin-like components	Purple to deep-purple cytoplasm	Clear positive color; can assist display of ground-glass cells	Stains multiple tissue components; requires strict localization
Victoria blue method	HBsAg-related cytoplasmic material and elastic fibers	Blue to blue-purple cytoplasm	Prominent display of cytoplasmic positive structures	Requires distinction between hepatocyte positivity and elastic fiber background

6 HBsAg Immunohistochemistry

6.1 Method principle

(1) Antigen-specific recognition

HBsAg immunohistochemistry uses specific antibodies against hepatitis B surface antigen to display the location of HBsAg expression in tissue sections. Compared with orcein, aldehyde fuchsin, and Victoria blue, this method has higher specificity and more direct value for viral antigen localization.

(2) Positive localization

HBsAg is mostly located in hepatocyte cytoplasm, but it can also show membranous, granular, or clumped distribution. Diffuse cytoplasmic positivity is often associated with ground-glass hepatocytes, while membranous positivity may suggest distribution of surface antigen near the cell membrane or membranous structures.

(3) Chromogenic system

When DAB chromogen is used, positive signals appear brownish-yellow to brownish-brown. After nuclear counterstaining, hepatic lobular architecture, cell location, and the distribution range of positive cells can be observed simultaneously.

6.2 Application features

(1) Advantages

HBsAg immunohistochemistry can directly demonstrate HBV surface antigen expression in liver tissue and is an important method for confirming HBV-related infection at the tissue level. It can distinguish cytoplasmic positivity, membranous positivity, and focal positivity in hepatocytes and is more reliable than traditional histochemical staining.

(2) Limitations

HBsAg positivity indicates the presence of surface antigen in tissue, but it does not by itself prove active viral replication. Some chronic carrier states or low-replication states may also show HBsAg positivity. Therefore, interpretation should be combined with HBcAg, HBV DNA, serological markers, and clinical background.

(3) Interpretation points

The proportion of positive cells, distribution area, staining intensity, and positivity pattern should be assessed. Diffuse cytoplasmic positivity, scattered positivity, and membranous positivity have different implications, but all require comprehensive interpretation with HBcAg and serological results.

7 HBcAg Immunohistochemistry

7.1 Method principle

(1) Core antigen detection

HBcAg immunohistochemistry uses antibodies against hepatitis B core antigen to detect HBcAg expression in hepatocytes. HBcAg is more closely related to viral replication status, so it has important auxiliary value in assessing viral activity.

(2) Positive localization patterns

HBcAg can appear as nuclear positivity, cytoplasmic positivity, or mixed nuclear-cytoplasmic positivity. Nuclear positivity often suggests the presence of core antigen in the nucleus; cytoplasmic positivity may be related to viral assembly, antigen transport, or replication-associated states.

(3) Complementarity with HBsAg

HBsAg and HBcAg reflect different viral components. HBsAg positivity more often indicates surface antigen expression or accumulation, while HBcAg positivity is more helpful for evaluating viral replication-related antigen status. Combined detection of both has greater interpretive value than either marker alone.

7.2 Application features

(1) Advantages

HBcAg immunohistochemistry can display core antigen localization on tissue sections and helps determine virus-related activity status. For cases in which serological and histological results are not fully consistent, HBcAg can provide important supplementary information.

(2) Limitations

HBcAg expression is affected by viral replication level, host immune status, and antiviral therapy. A negative result does not completely exclude HBV infection, especially in low-replication, post-treatment, or poorly preserved samples. HBsAg and HBV DNA should be used together for interpretation.

(3) Interpretation points

Nuclear positivity, cytoplasmic positivity, and mixed positivity should be distinguished, and the distribution of positive cells should be recorded. Scattered weak positivity alone should be interpreted cautiously, while strong or widespread positivity usually has greater pathological reference value.

Table 3 Comparison of HBsAg and HBcAg immunohistochemistry

Item	HBsAg immunohistochemistry	HBcAg immunohistochemistry
Detection target	Hepatitis B surface antigen	Hepatitis B core antigen
Common localization	Cytoplasmic, membranous, granular	Nuclear, cytoplasmic, or mixed nuclear-cytoplasmic
Main significance	Shows surface antigen expression and accumulation	Helps assess viral replication-related antigen status
Relationship with ground-glass hepatocytes	Closely related	Ground-glass change is not the main feature
Specificity	High	High
Limitation	Cannot independently determine replication activity	Negative result cannot completely exclude HBV infection

8 HBV In Situ Hybridization

8.1 Method principle

(1) Spatial localization of nucleic acids

HBV in situ hybridization detects HBV DNA or RNA signals in tissue sections using specific probes. It can display the cells and regions containing viral nucleic acids while preserving tissue architecture. This method is suitable for studying viral nucleic acid distribution and localization of infected cells in tissue.

(2) Differences in detection targets

Detection of HBV DNA is more oriented toward the presence of viral genome or replication-associated nucleic acids. Detection of HBV RNA can reflect transcriptional activity or viral RNA expression. Different probe designs correspond to different interpretive directions and should not be treated as the same indicator.

(3) Signal interpretation

Positive signals may be located in the nucleus, cytoplasm, or both, depending on the detection target, probe type, and viral life cycle status. Interpretation should be combined with positive controls, negative probe controls, and antigen detection results.

8.2 Application features

(1) Advantages

In situ hybridization has high specificity and provides spatial localization information. It is valuable for studying the distribution of viral nucleic acids in different hepatic lobular regions, different cell populations, or different lesion areas.

(2) Limitations

This method requires high-quality tissue fixation, nucleic acid preservation, probe quality, and background control. It is more complex than routine immunohistochemistry. In routine clinical pathology screening, HBsAg and HBcAg immunohistochemistry are usually more commonly used.

(3) Applicable scenarios

It is suitable for supplementary validation in difficult cases, viral replication studies, observation of changes in intra-tissue viral nucleic acids before and after antiviral therapy, and spatial localization analysis when immunohistochemistry results are inconsistent.

9 Method Selection and Combined Interpretation

9.1 Method selection for different experimental objectives

(1) Routine pathological diagnosis

HE is used to assess hepatitis activity and tissue injury background. HBsAg and HBcAg immunohistochemistry are used to confirm HBV antigen expression in tissue. These form the core combination in histological evaluation.

(2) Traditional histochemical screening

Orcein, aldehyde fuchsin, and Victoria blue staining can be used to display HBsAg-related cytoplasmic changes, but they should not be used as independent diagnostic evidence. They are more suitable for teaching, traditional method comparison, and auxiliary identification of ground-glass hepatocytes.

(3) Viral nucleic acid localization research

If the question focuses on whether HBV DNA/RNA is present in specific cells or regions, in situ hybridization should be selected. If routine confirmation of HBV-related antigen expression is needed, immunohistochemistry is more practical.

Table 4 Selection of HBV histological detection methods

Experimental objective	Recommended method	Reason for selection	Notes
Assess hepatitis activity	HE staining	Shows inflammation, necrosis, and tissue architecture	Cannot directly prove HBV infection
Identify ground-glass hepatocytes	HE + HBsAg immunohistochemistry	Combines morphology and antigen localization	HE suggests, IHC confirms
Traditional display of HBsAg-related cytoplasmic changes	Orcein, aldehyde fuchsin, Victoria blue	Can assist display of cytoplasmic inclusions	Less specific than immunohistochemistry
Confirm HBsAg expression	HBsAg immunohistochemistry	Direct localization of surface antigen	Cannot independently determine replication activity
Evaluate core antigen expression	HBcAg immunohistochemistry	Shows core antigen localization	Requires combination with HBV DNA and serology
Localize viral nucleic acids	HBV in situ hybridization	Shows spatial distribution of DNA/RNA	Requires high sample quality and probe performance

9.2 Interpretation of common result combinations

(1) HE-suspected ground-glass change with HBsAg positivity

This combination supports HBsAg accumulation in hepatocytes and is a relatively typical pattern of HBV-related liver tissue change. If HBcAg is also positive, serum HBV DNA should be used to further evaluate viral activity.

(2) Traditional histochemical positivity with negative HBsAg immunohistochemistry

Nonspecific staining, copper-binding proteins, or interference from other cytoplasmic components should be considered. In this situation, HBV infection should not be judged solely based on orcein, aldehyde fuchsin, or Victoria blue positivity.

(3) HBsAg positivity with HBcAg negativity

This suggests the presence of surface antigen expression or accumulation in tissue, but core antigen is not obvious. It may be seen in low-replication states, post-treatment states, or cases with low HBcAg expression. Serology and molecular results should be combined.

(4) HBcAg positivity with weak or negative HBsAg

This situation requires review of technical quality, antibody performance, tissue preservation, and serological results. HBV DNA/RNA in situ hybridization or molecular detection may be added if necessary.

Table 5 Combined interpretation of HBV histological staining results

Result combination	Possible implication	Recommended supplement
HE ground-glass appearance + HBsAg positivity	HBsAg-related cytoplasmic accumulation	Combine with HBcAg and serum HBV DNA
Orcein positivity + HBsAg positivity	Traditional staining and antigen localization are consistent	Can support HBV-related tissue changes
Orcein positivity + HBsAg negativity	Possible nonspecific staining	Recheck antibody, controls, and copper-related deposition
HBsAg positivity + HBcAg positivity	Both surface antigen and core antigen are expressed	Combine with viral load and inflammatory activity
HBsAg positivity + HBcAg negativity	Mainly surface antigen expression	Combine with treatment history, serology, and molecular results
IHC negativity + HBV in situ hybridization positivity	Nucleic acid signal may be present but antigen expression is weak	Recheck probe, antibody, and sample quality
HE chronic hepatitis changes + HBV marker negativity	Tissue morphology alone does not support HBV diagnosis	Evaluate HCV, autoimmune hepatitis, drug-induced liver injury, etc.

10 Selection of Related Reagents and Materials

Table 6 Key reagents and materials for HBV histological staining

Product/Material Name	CAS No.	Product Category	Application Positioning
Hematoxylin	517-28-2	HE staining/nuclear staining reagent	Used for HE staining and immunohistochemical counterstaining to display nuclei and tissue architecture
Eosin Y	17372-87-1	HE staining reagent	Used in HE staining to display cytoplasm, red blood cells, and tissue background
Orcein	1400-62-0	Histochemical dye	Used in the orcein method to assist display of HBsAg-related cytoplasmic changes
Basic fuchsin	632-99-5	Aldehyde fuchsin-related staining component	Used in aldehyde fuchsin staining systems to assist display of cytoplasmic inclusions and related structures
Victoria blue B	2580-56-5	Histochemical dye	Used in the Victoria blue method to display HBsAg-related cytoplasmic material and elastic structures
DAB	91-95-2	Immunohistochemical chromogenic substrate	Used for HRP system chromogenic detection, producing brownish-yellow to brownish-brown positive signals
Citric acid	77-92-9	Antigen retrieval buffer component	Used in heat-induced antigen retrieval systems for immunohistochemistry
Disodium EDTA	6381-92-6	Antigen retrieval buffer component	Used in some antigen retrieval systems to improve antibody binding

Table 7 Product selection for HBV histological staining, immunohistochemistry, and supporting detection

Cat. No.	Product Name	Grade/Specification	Product Category	Application Positioning
H1510444	Hepatitis B Virus Staining Solution (Shikata’s Orcein Method)	BioReagent,Biological Stain,Suitable for microbiology,for microscopy	HBV traditional histochemical staining solution	Used in the Shikata orcein method to assist display of HBsAg-related cytoplasmic inclusions in hepatocytes; suitable for auxiliary identification of ground-glass hepatocytes
H1510443	Hepatitis B Virus Staining Solution (Tanaka Victoria Blue Method)	BioReagent,Suitable for microbiology,for microscopy,Biological Stain	HBV traditional histochemical staining solution	Used in the Tanaka Victoria blue method to assist display of HBsAg-related cytoplasmic material; suitable for comparative interpretation with HE and HBsAg immunohistochemistry
D1505853	DAB Chromogenic Reagent Kit	BioReagent,chromogenic agent,Suitable for Immunohistochemistry(IHC),for microscopy	IHC chromogenic reagent	Used for HRP-system chromogenic detection in HBsAg, HBcAg, and other immunohistochemistry, producing brownish-yellow to brownish-brown positive signals
D1374036	DAB Horseradish Peroxidase Color Development Enhancer (100×)	BioReagent,Suitable for Immunohistochemistry(IHC),100×	IHC chromogenic enhancer	Used to enhance DAB chromogenic signals; suitable for optimizing weakly positive HBV antigen immunohistochemistry results
E745530	EDTA Antigen Retrieval Solution (50X)	BioReagent, sterile-filtered, Suitable for Immunohistochemistry(IHC), sterile, 50X	Antigen retrieval solution	Used for antigen exposure in paraffin-section HBsAg/HBcAg immunohistochemistry to improve antibody binding efficiency
T1508979	Tris-EDTA Antigen Retrieval Solution (10×, pH 8.0)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for heat-induced retrieval of HBV antigen immunohistochemistry under medium-to-high pH conditions
T1508980	Tris-EDTA Antigen Retrieval Solution (10×, pH 9.0)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for optimization of medium-to-high pH antigen retrieval conditions in HBsAg/HBcAg immunohistochemistry
T1508981	Tris-EDTA Antigen Retrieval Solution (10×, pH 9.5)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for higher-pH antigen retrieval systems; suitable for IHC optimization under different antibody clones and fixation conditions
T751378	Tris-EDTA Antigen Retrieval Solution (50×)	BioReagent, Suitable for Immunohistochemistry(IHC), 50×	Antigen retrieval solution	Used for preparing Tris-EDTA antigen retrieval solution in batch IHC experiments
T1508976	Tris Antigen Retrieval Solution (10×, pH 9.0)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for screening alkaline retrieval conditions in HBV antigen immunohistochemistry
T1508977	Tris Antigen Retrieval Solution (10×, pH 9.5)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for IHC retrieval optimization in heavily fixed or insufficiently exposed antigen samples
T1508978	Tris Antigen Retrieval Solution (10×, pH 10.0)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC),10×	Antigen retrieval solution	Used for high-pH antigen retrieval schemes, suitable for exploring some antibody conditions
I752100	Improved Citrate Antigen Retrieval Solution (50×)	BioReagent, Suitable for Immunohistochemistry(IHC), 50X	Antigen retrieval solution	Used for heat retrieval in citrate systems; suitable for routine optimization of HBV antigen IHC conditions
C752119	Citrate Antigen Retrieval Solution (50X)	BioReagent, sterile-filtered, Suitable for Immunohistochemistry(IHC), sterile, 50X	Antigen retrieval solution	Used for routine antigen retrieval in paraffin-section immunohistochemistry
C752118	Citrate-EDTA Antigen Retrieval Solution (40X)	BioReagent, sterile-filtered, sterile, Suitable for Immunohistochemistry(IHC), 40X	Antigen retrieval solution	Used in citrate and EDTA combined retrieval systems; suitable for screening different HBV antigen-antibody combinations
P1516096	Paraffin Section Deparaffinization Antigen Retrieval Solution (20×, Low pH)	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)	Deparaffinization antigen retrieval solution	Used for paraffin section deparaffinization and low-pH antigen retrieval; suitable for integration into IHC workflows
W1509002	Paraffin Section Deparaffinization Antigen Retrieval Solution (20×, High pH)	Suitable for microbiology,for microscopy,BioReagent	Deparaffinization antigen retrieval solution	Used for paraffin section deparaffinization and high-pH antigen retrieval; suitable for optimizing HBsAg/HBcAg IHC conditions
A752145	All-purpose Powerful Antigen Retrieval Solution (10×)	BioReagent, Suitable for Immunohistochemistry(IHC), 10X	Strong antigen retrieval solution	Used for immunohistochemical retrieval optimization in heavily fixed or weakly exposed tissues
Q752080	Quick Antigen Retrieval Solution for Frozen Sections (5X)	sterile-filtered, BioReagent, Suitable for Immunohistochemistry(IHC), sterile, 5X	Frozen-section antigen retrieval solution	Used for pretreatment before HBV antigen immunostaining in frozen liver tissue sections
G1508982	Pepsin Antigen Retrieval Solution	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)	Enzymatic antigen retrieval solution	Used for protease digestion-based antigen retrieval; suitable for IHC optimization under certain antibody or fixation conditions
P1509544	Pepsin Antigen Retrieval Kit	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)	Enzymatic antigen retrieval kit	Used for standardized pepsin retrieval workflows and auxiliary screening of HBV antigen IHC conditions
P1508983	Trypsin Antigen Retrieval Solution	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)	Enzymatic antigen retrieval solution	Used for trypsin digestion retrieval, suitable for samples with insufficient antigen exposure
T1509543	Trypsin Antigen Retrieval Kit	BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)	Enzymatic antigen retrieval kit	Used for standardized trypsin retrieval workflows and auxiliary immunohistochemistry method optimization
A752121	Antigen Retrieval Solution for Floating Sections (10×)	BioReagent, sterile-filtered, Suitable for Immunohistochemistry(IHC), 10X	Floating-section antigen retrieval solution	Used for antigen retrieval during floating section processing and suitable for specific IHC workflow optimization
N116470	Neutral gum	FMP	Mounting material	Used for mounting, transparent preservation, and microscopic observation of HBV histochemical and immunohistochemical sections
EJ1513766	Human Hepatitis B Virus Surface Antigen (HBsAg) ELISA Kit	BioReagent	HBsAg ELISA detection product	Used for quantitative or semi-quantitative detection of HBsAg in human samples; can serve as supporting validation for histological HBsAg staining results
EJ1512618	Mouse Hepatitis B Surface Antigen (HBsAg) ELISA Kit	BioReagent	HBsAg ELISA detection product	Used for HBsAg detection in mouse model samples; suitable for supporting analysis of tissue staining results in HBV animal models
EJ1513764	Human Hepatitis B core Antigen (HBcAg) ELISA Kit	BioReagent	HBcAg ELISA detection product	Used for HBcAg detection in human samples; can assist interpretation of HBcAg immunohistochemistry results and viral antigen status
EJ1511961	Rat Hepatitis B Virus Core Antigen(HBcAg) ELISA Kit	BioReagent	HBcAg ELISA detection product	Used for HBcAg detection in rat model samples; suitable for supporting validation of histological results in animal experiments
EJ1512617	Mouse Hepatitis B Virus Core Antigen (HBcAg) ELISA Kit	BioReagent	HBcAg ELISA detection product	Used for HBcAg detection in mouse model samples; assists analysis of tissue antigen results in HBV infection models
EJ1513765	Human Hepatitis B Virus Core Antigen-Associated Antigen (HBcrAg) ELISA Kit	BioReagent	HBcrAg ELISA detection product	Used for HBV core-related antigen detection; can serve as a supplementary indicator of viral replication-related status and tissue HBcAg results
EJ1513763	Human Hepatitis B Surface Antigen Large Protein (HBV-LP) ELISA Kit	BioReagent	HBV large protein ELISA detection product	Used for HBV large protein detection; suitable for joint analysis with HBsAg tissue localization and serum/body fluid detection results

11 Common Questions

11.1 Can HE staining diagnose hepatitis B virus infection?

No. HE staining can only show liver tissue injury patterns and suggestive changes such as ground-glass hepatocytes. It cannot directly prove HBV infection. HBsAg and HBcAg immunohistochemistry should be used to confirm HBV-related antigen expression in tissue.

11.2 Does orcein positivity equal HBsAg positivity?

Not completely. Orcein can display HBsAg-related cytoplasmic material, but it may also show copper-binding proteins or other cytoplasmic components. Orcein positivity should be regarded as an auxiliary clue and cannot replace HBsAg immunohistochemistry.

11.3 Which is more important, HBsAg immunohistochemistry or HBcAg immunohistochemistry?

They serve different purposes. HBsAg immunohistochemistry is used to display surface antigen expression and accumulation, while HBcAg immunohistochemistry is more helpful for evaluating core antigen expression and viral replication-related status. In practice, combined use is usually recommended.

11.4 Can HBcAg negativity exclude HBV infection?

No. HBcAg expression may be affected by viral replication level, immune status, treatment, and tissue preservation conditions. When HBcAg is negative, interpretation should still include HBsAg, serum HBV markers, and HBV DNA results.

11.5 Is in situ hybridization more suitable than immunohistochemistry for routine screening?

Not necessarily. In situ hybridization is suitable for viral nucleic acid localization and research use, but it is more complex and requires higher sample quality. In routine pathology, HBsAg and HBcAg immunohistochemistry are usually more practical.

11.6 Are traditional histochemical stains still necessary?

Traditional histochemical stains can no longer replace immunohistochemistry and molecular detection for routine confirmation of HBV infection. However, orcein, aldehyde fuchsin, and Victoria blue can still be used for historical case comparison, teaching, auxiliary identification of ground-glass hepatocytes, and methodological comparison.

Histological staining for identifying hepatitis B virus should use HE to evaluate the tissue injury background, HBsAg and HBcAg immunohistochemistry to confirm viral antigen expression, and HBV DNA/RNA in situ hybridization when nucleic acid localization is needed. Orcein, aldehyde fuchsin, and Victoria blue methods can serve as traditional auxiliary methods, but they cannot be used alone as confirmatory evidence of HBV infection.

For more related articles, please see below:

[1] Clinical manifestations and specimen collection of hepatitis viruses

[2] Hepatitis virus prevention and treatment

[3] Hepatitis A virus RNA extraction